Secreted proteins and uses thereof

ABSTRACT

The invention provides isolated nucleic acid molecules, designated TANGO 228 nucleic acid molecules, which encode secreted proteins with homology to the rat MCA-32 protein, isolated nucleic acid molecules, designated TANGO 240 nucleic acid molecules, which encode secreted proteins with homology to the  Mycobacterium tuberculosis  hypothetical protein Rv0712, and isolated nucleic acid molecules, designated TANGO 243 nucleic acid molecules, which encode proteins with homology to human PLAP (phospholipase A2-activating protein). 
     The invention also provides antisense nucleic acid molecules, expression vectors containing the nucleic acid molecules of the invention, host cells into which the expression vectors have been introduced, and non-human transgenic animals in which a nucleic acid molecule of the invention has been introduced or disrupted. The invention still further provides isolated polypeptides, fusion polypeptides, antigenic peptides and antibodies. Diagnostic, screening and therapeutic methods utilizing compositions of the invention are also provided.

BACKGROUND OF THE INVENTION

Many secreted proteins, for example, cytokines and cytokine receptors,play a vital role in the regulation of cell growth, celldifferentiation, and a variety of specific cellular responses. A numberof medically useful proteins, including erythropoietin,granulocyte-macrophage colony stimulating factor, human growth hormone,and various interleukins, are secreted proteins. Thus, an important goalin the design and development of new therapies is the identification andcharacterization of secreted and transmembrane proteins and the geneswhich encode them.

Many secreted proteins are receptors which bind a ligand and transducean intracellular signal, leading to a variety of cellular responses. Theidentification and characterization of such a receptor enables one toidentify both the ligands which bind to the receptor and theintracellular molecules and signal transduction pathways associated withthe receptor, permitting one to identify or design modulators ofreceptor activity, e.g., receptor agonists or antagonists and modulatorsof signal transduction.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery ofcDNA molecules which encode the TANGO 228, 240, and 243 proteins, all ofwhich are either wholly secreted or transmembrane proteins.

The TANGO 228 proteins are homologous to rat surface protein MCA-32(mast cell Ag-32), a component of the immunologic pathway.

The TANGO 240 proteins are homologous to the Mycobacterium tuberculosisconserved hypothetical protein Rv0712.

The TANGO 243 proteins share significant homology to human PLAP(phospholipase A2-activating protein), a modulator of arthropathicdisorders.

The TANGO 228, TANGO 240, and TANGO 243 proteins, fragments,derivatives, and variants thereof are collectively referred to herein as“polypeptides of the invention” or “proteins of the invention.” Nucleicacid molecules encoding the polypeptides or proteins of the inventionare collectively referred to as “nucleic acids of the invention.”

The nucleic acids and polypeptides of the present invention are usefulas modulating agents in regulating a variety of cellular processes.Accordingly, in one aspect, this invention provides isolated nucleicacid molecules encoding a polypeptide of the invention or a biologicallyactive portion thereof. The present invention also provides nucleic acidmolecules which are suitable for use as primers or hybridization probesfor the detection of nucleic acids encoding a polypeptide of theinvention.

The invention features nucleic acid molecules which are at least 30% (or35%, 40%, 45%, 50%, 55%, 65%, 75%, 85%, 95%, or 98%) identical to thenucleotide sequence of SEQ ID NO:1, the nucleotide sequence of the cDNAinsert of a clone deposited with ATCC as Accession Number 207116, or acomplement thereof.

The invention features nucleic acid molecules which are at least 48% (or50%, 55%, 60%, 65%, 75%, 85%, 95%, or 98%) identical to the nucleotidesequence of SEQ ID NO:2, the nucleotide sequence of the cDNA insert of aclone deposited with ATCC as Accession Number 207116, or a complementthereof.

The invention features nucleic acid molecules which are at least 30% (or40%, 45%, 50%, 55%, 65%, 75%, 85%, 95%, or 98%) identical to thenucleotide sequence of SEQ ID NO:13 or 14, the nucleotide sequence ofthe cDNA insert of a clone deposited with ATCC as Accession Number207116, or a complement thereof.

The invention features nucleic acid molecules which are at least 80% (or82%, 85%, 87%, 90%, 92%, 95%, or 98%) identical to the nucleotidesequence of SEQ ID NO:19, the nucleotide sequence of the cDNA insert ofa clone deposited with ATCC as Accession Number 207116, or a complementthereof.

The invention features nucleic acid molecules which are at least 93% (or94%, 95%, 96%, 97%, or 98%) identical to the nucleotide sequence of SEQID NO:20, the nucleotide sequence of the cDNA insert of a clonedeposited with ATCC as Accession Number 207116, or a complement thereof.

The invention features nucleic acid molecules which include a fragmentof at least 310 (400, 500, 600, 800, 1000, 1250, 1500, 1750, 2000, 2250,2500, 2750, 3000, 3250, 3500, 3750, 4000, or 4020) nucleotides of thenucleotide sequence of SEQ ID NO:1 the nucleotide sequence of the cDNAof ATCC Accession Number 207116, or a complement thereof.

The invention features nucleic acid molecules which are at least 30% (or40%, 45%, 50%, 55%, 65%, 75%, 85%, 95%, or 98%) identical to thenucleotide sequence of SEQ ID NO:31 or 32, or a complement thereof.

The invention features nucleic acid molecules which are at least 30% (or40%, 45%, 50%, 55%, 65%, 75%, 85%, 95%, or 98%) identical to thenucleotide sequence of SEQ ID NO:39 or 40, or a complement thereof.

The invention features nucleic acid molecules which include a fragmentof at least 515 (530, 550, 600, 700, 800, 900, 1000, 1100, 1200, 1300,1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, or 2150) nucleotides ofthe nucleotide sequence of SEQ ID NO:13, the nucleotide sequence of thecDNA of ATCC Accession Number 207116, or a complement thereof.

The invention features nucleic acid molecules which include a fragmentof at least 2220 (2260, 2300, 2340, 2380, 2420, 2460, 2500, 2540, 2580,2620, 2660, 2700, 2740, 2780, or 2800) nucleotides of the nucleotidesequence of SEQ ID NO:19, the nucleotide sequence of the cDNA of ATCCAccession Number 207116, or a complement thereof.

The invention features nucleic acid molecules which include a fragmentof at least 50 (100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600,650, 700, 750, 800, or 900) nucleotides of the nucleotide sequence ofSEQ ID NO:31 or 32, or a complement thereof.

The invention features nucleic acid molecules which include a fragmentof at least 50 (100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600,700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800,1900, 2000, 2100, or 220) nucleotides of the nucleotide sequence of SEQID NO:39 or 40, or a complement thereof.

The invention also features nucleic acid molecules which include anucleotide sequence encoding a protein having an amino acid sequencethat is at least 30% (or 35%, 40%, 45%, 50%, 55%, 60%, 65%, 75%, 85%,95%, or 98%) identical to the amino acid sequence of SEQ ID NO:3, theamino acid sequence encoded by the cDNA of ATCC Accession Number 207116,or a complement thereof.

The invention also features nucleic acid molecules which include anucleotide sequence encoding a protein having an amino acid sequencethat is at least 35% (or 40%, 45%, 50%, 55%, 65%, 75%, 85%, 95%, or 98%)identical to the amino acid sequence of SEQ ID NO:15, the amino acidsequence encoded by the cDNA of ATCC Accession Number 207116, or acomplement thereof.

The invention also features nucleic acid molecules which include anucleotide sequence encoding a protein having an amino acid sequencethat is at least 93% (or 94%, 95%, 96%, 97% or 98%) identical to theamino acid sequence of SEQ ID NO:21, the amino acid sequence encoded bythe cDNA of ATCC Accession Number 207116, or a complement thereof.

The invention also features nucleic acid molecules which include anucleotide sequence encoding a protein having an amino acid sequencethat is at least 35% (or 40%, 45%, 50%, 55%, 65%, 75%, 85%, 95%, or 98%)identical to the amino acid sequence of SEQ ID NO:33, the amino acidsequence encoded by the cDNA of ATCC Accession Number 207116, or acomplement thereof.

The invention also features nucleic acid molecules which include anucleotide sequence encoding a protein having an amino acid sequencethat is at least 35% (or 40%, 45%, 50%, 55%, 65%, 75%, 85%, 95%, or 98%)identical to the amino acid sequence of SEQ ID NO:41, the amino acidsequence encoded by the cDNA of ATCC Accession Number 207116, or acomplement thereof.

The invention also features nucleic acid molecules which include anucleotide sequence encoding a protein having an amino acid sequencethat is at least 35% (or 40%, 45%, 50%, 55%, 65%, 75%, 85%, 95%, or 98%)identical to the amino acid sequence of SEQ ID NO:45, the amino acidsequence encoded by the cDNA of ATCC Accession Number 207116, or acomplement thereof.

In preferred embodiments, the nucleic acid molecules have the nucleotidesequence of SEQ ID NO:1, 2, 13, 14, 19, 20, 31, 32, 39, 40, 44 or thenucleotide sequence of the cDNA of ATCC Accession Number 207116.

Also within the invention are nucleic acid molecules which encode afragment of a polypeptide having the amino acid sequence of SEQ ID NO:3,or a fragment including at least 15 (25, 30, 50, 75, 100, 125, 150, 175,200, 225, 250, 275, 300, or 340) contiguous amino acids of SEQ ID NO:3,or the amino acid sequence encoded by the cDNA of ATCC Accession Number207116.

Also within the invention are nucleic acid molecules which encode afragment of a polypeptide having the amino acid sequence of SEQ IDNO:15, or a fragment including at least 15 (25, 30, 50, 75, 100, 125,150, 175, 200, 225, 250, 275, 300, 325, 350, 360, or 370) contiguousamino acids of SEQ ID NO:15, or the amino acid sequence encoded by thecDNA of ATCC Accession Number 207116.

Also within the invention are nucleic acid molecules which encode afragment of a polypeptide having the amino acid sequence of SEQ IDNO:21, or a fragment including at least 740 (745, 750, 755, 760, 765,770, 775, 780, 785, or 790) contiguous amino acids of SEQ ID NO:21, orthe amino acid sequence encoded by the cDNA of ATCC Accession Number207116.

Also within the invention are nucleic acid molecules which encode afragment of a polypeptide having the amino acid sequence of SEQ IDNO:33, or a fragment including at least 15 (25, 30, 50, 75, 100, 125,150, 175, 200, 225, or 240) contiguous amino acids of SEQ ID NO:33.

Also within the invention are nucleic acid molecules which encode afragment of a polypeptide having the amino acid sequence of SEQ IDNO:41, or a fragment including at least 15 (25, 30, 50, 75, 100, 125,150, 175, 200, 250, 300, 350 or 370) contiguous amino acids of SEQ IDNO:41.

Also within the invention are nucleic acid molecules which encode afragment of a polypeptide having the amino acid sequence of SEQ IDNO:21, or a fragment including at least 740 (745, 750, 755, 760, 765,770, 775, 780, 785, 790, 800, 825, 850, or 880) contiguous amino acidsof SEQ ID NO:21, or the amino acid sequence encoded by the cDNA of ATCCAccession Number 207116.

The invention includes nucleic acid molecules which encode a naturallyoccurring allelic variant of a polypeptide comprising the amino acidsequence of SEQ ID NO:3, 15, 21, 33, 41, or 45, or the amino acidsequence encoded by the cDNA of ATCC Accession Number 207116, whereinthe nucleic acid molecule hybridizes to a nucleic acid moleculeconsisting of a nucleic acid sequence encoding SEQ ID NO:3, 15, 21, 33,41, or 45, the amino acid sequence encoded by the cDNA of ATCC AccessionNumber 207116, or a complement thereof under stringent conditions.

Also within the invention are isolated polypeptides or proteins havingan amino acid sequence that is at least about 30%, preferably 35%, 45%,55%, 65%, 75%, 85%, 95%, or 98% identical to the amino acid sequence ofSEQ ID NO:3, or the amino acid sequence encoded by the cDNA of ATCCAccession Number 207116.

Also within the invention are isolated polypeptides or proteins havingan amino acid sequence that is at least about 35%, preferably 40%, 45%,50%, 55%, 65%, 75%, 85%, 95%, or 98% identical to the amino acidsequence of SEQ ID NO:15, or the amino acid sequence encoded by the cDNAof ATCC Accession Number 207116.

Also within the invention are isolated polypeptides or proteins havingan amino acid sequence that is at least about 93%, preferably 94%, 95%,96%, 97% or 98% identical to the amino acid sequence of SEQ ID NO:21, orthe amino acid sequence encoded by the cDNA of ATCC Accession Number207116.

Also within the invention are isolated polypeptides or proteins havingan amino acid sequence that is at least about 35%, preferably 40%, 45%,50%, 55%, 65%, 75%, 85%, 95%, or 98% identical to the amino acidsequence of SEQ ID NO:33.

Also within the invention are isolated polypeptides or proteins havingan amino acid sequence that is at least about 35%, preferably 40%, 45%,50%, 55%, 65%, 75%, 85%, 95%, or 98% identical to the amino acidsequence of SEQ ID NO:41

Also within the invention are isolated polypeptides or proteins havingan amino acid sequence that is at least about 35%, preferably 40%, 45%,50%, 55%, 65%, 75%, 85%, 95%, or 98% identical to the amino acidsequence of SEQ ID NO:45.

Also within the invention are isolated polypeptides or proteins whichare encoded by a nucleic acid molecule having a nucleotide sequence thatis at least about 48%, preferably 50%, 55%, 60%, 65%, 75%, 85%, or 95%identical to the nucleic acid sequence encoding SEQ ID NO:3, andisolated polypeptides or proteins which are encoded by a nucleic acidmolecule having a nucleotide sequence which hybridizes under stringenthybridization conditions to a nucleic acid molecule having thenucleotide sequence of SEQ ID NO:1 or 2, a complement thereof, or thenon-coding strand of the cDNA of ATCC Accession Number 207116.

Also within the invention are isolated polypeptides or proteins whichare encoded by a nucleic acid molecule having a nucleotide sequence thatis at least about 30%, preferably 35%, 40%, 45%, 50%, 55%, 60%, 65%,75%, 85%, or 95% identical to the nucleic acid sequence encoding SEQ IDNO:15, and isolated polypeptides or proteins which are encoded by anucleic acid molecule having a nucleotide sequence which hybridizesunder stringent hybridization conditions to a nucleic acid moleculehaving the nucleotide sequence of SEQ ID NO:13 or 14, a complementthereof, or the non-coding strand of the cDNA of ATCC Accession Number207116.

Also within the invention are isolated polypeptides or proteins whichare encoded by a nucleic acid molecule having a nucleotide sequence thatis at least about 93%, preferably 94%, 95%, 96%, 97%, or 98% identicalto the nucleic acid sequence encoding SEQ ID NO:21, and isolatedpolypeptides or proteins which are encoded by a nucleic acid moleculehaving a nucleotide sequence which hybridizes under stringenthybridization conditions to a nucleic acid molecule having thenucleotide sequence of SEQ ID NO:19, 20, or 44, a complement thereof, orthe non-coding strand of the cDNA of ATCC Accession Number 207116.

Also within the invention are isolated polypeptides or proteins whichare encoded by a nucleic acid molecule having a nucleotide sequence thatis at least about 30%, preferably 35%, 40%, 45%, 50%, 55%, 60%, 65%,75%, 85%, or 95% identical to the nucleic acid sequence encoding SEQ IDNO:33, and isolated polypeptides or proteins which are encoded by anucleic acid molecule having a nucleotide sequence which hybridizesunder stringent hybridization conditions to a nucleic acid moleculehaving the nucleotide sequence of SEQ ID NO:31 or 32, a complementthereof.

Also within the invention are isolated polypeptides or proteins whichare encoded by a nucleic acid molecule having a nucleotide sequence thatis at least about 30%, preferably 35%, 40%, 45%, 50%, 55%, 60%, 65%,75%, 85%, or 95% identical to the nucleic acid sequence encoding SEQ IDNO:41, and isolated polypeptides or proteins which are encoded by anucleic acid molecule having a nucleotide sequence which hybridizesunder stringent hybridization conditions to a nucleic acid moleculehaving the nucleotide sequence of SEQ ID NO:39 or 40.

Also within the invention are isolated polypeptides or proteins whichare encoded by a nucleic acid molecule having a nucleotide sequence thatis at least about 30%, preferably 35%, 40%, 45%, 50%, 55%, 60%, 65%,75%, 85%, or 95% identical to the nucleic acid sequence encoding SEQ IDNO:45, and isolated polypeptides or proteins which are encoded by anucleic acid molecule having a nucleotide sequence which hybridizesunder stringent hybridization conditions to a nucleic acid moleculehaving the nucleotide sequence of SEQ ID NO:19 or 44.

Also within the invention are polypeptides which are naturally occurringallelic variants of a polypeptide that includes the amino acid sequenceof SEQ ID NO:3, 15, 21, 33, 41, or 45, or the amino acid sequenceencoded by the cDNA of ATCC Accession Number 207116, wherein thepolypeptide is encoded by a nucleic acid molecule which hybridizes to anucleic acid molecule having the sequence of SEQ ID NO:1, 2, 3, 13, 14,19, 20, 31, 32, 39, 40, or 44 or a complement thereof under stringentconditions.

The invention also features nucleic acid molecules that hybridize understringent conditions to a nucleic acid molecule having the nucleotidesequence of SEQ ID NO:1 or 2, the cDNA of ATCC Accession Number 207116,or a complement thereof. In other embodiments, the nucleic acidmolecules are at least 310 (400, 500, 600, 800, 1000, 1250, 1500, 1750,2000, 2250, 2500, 2750, 3000, 3250, 3500, 3750, 4000, or 4020)nucleotides in length and hybridize under stringent conditions to anucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1or 2, the cDNA of ATCC Accession Number 207116, or a complement thereof.

The invention also features nucleic acid molecules that hybridize understringent conditions to a nucleic acid molecule having the nucleotidesequence of SEQ ID NO:13 or 14, the cDNA of ATCC Accession Number207116, or a complement thereof. In other embodiments, the nucleic acidmolecules are at least 515 (530, 550, 600, 700, 800, 900, 1000, 1100,1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, or 2150)nucleotides in length and hybridize under stringent conditions to anucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:13or 14, the cDNA of ATCC Accession Number 207116, or a complementthereof.

The invention also features nucleic acid molecules that hybridize understringent conditions to a nucleic acid molecule having the nucleotidesequence of SEQ ID NO:19 or 20, or the cDNA of ATCC Accession Number207116, or a complement thereof. In other embodiments, the nucleic acidmolecules are at least 2220 (2260, 2300, 2340, 2380, 2420, 2460, 2500,2540, 2580, 2620, 2660, 2700, 2740, 2780, or 2800) nucleotides in lengthand hybridize under stringent conditions to a nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO:19 or 20, the cDNA ofATCC Accession Number 207116, or a complement thereof.

The invention also features nucleic acid molecules that hybridize understringent conditions to a nucleic acid molecule having the nucleotidesequence of SEQ ID NO:31 or 32, or a complement thereof. In otherembodiments, the nucleic acid molecules are at least 100 (150, 200, 250,300, 350, 400, 500, 600, or 700) nucleotides in length and hybridizeunder stringent conditions to a nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO:31 or 32, or a complement thereof.

The invention also features nucleic acid molecules that hybridize understringent conditions to a nucleic acid molecule having the nucleotidesequence of SEQ ID NO:39 or 40, or a complement thereof. In otherembodiments, the nucleic acid molecules are at least 515 (530, 550, 600,700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800,1900, 2000, 2100, or 2150) nucleotides in length and hybridize understringent conditions to a nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO:39 or 40, or a complement thereof.

The invention also features nucleic acid molecules that hybridize understringent conditions to a nucleic acid molecule having the nucleotidesequence of SEQ ID NO:19 or 44, the cDNA of ATCC Accession Number207116, or a complement thereof. In other embodiments, the nucleic acidmolecules are at least 515 (530, 550, 600, 700, 800, 900, 1000, 1100,1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, or 2150)nucleotides in length and hybridize under stringent conditions to anucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:19or 44, the cDNA of ATCC Accession Number 207116, or a complementthereof.

In one embodiment, the invention provides an isolated nucleic acidmolecule which is antisense to the coding strand of a nucleic acid ofthe invention.

Another aspect of the invention provides vectors, e.g., recombinantexpression vectors, comprising a nucleic acid molecule of the invention.In another embodiment, the invention provides host cells containing sucha vector or a nucleic acid molecule of the invention. The invention alsoprovides methods for producing a polypeptide of the invention byculturing, in a suitable medium, a host cell of the invention containinga recombinant expression vector such that a polypeptide is produced.

Another aspect of this invention features isolated or recombinantproteins and polypeptides of the invention. Preferred proteins andpolypeptides possess at least one biological activity possessed by thecorresponding naturally-occurring human polypeptide. An activity, abiological activity, or a functional activity of a polypeptide ornucleic acid of the invention refers to an activity exerted by aprotein, polypeptide or nucleic acid molecule of the invention on aresponsive cell as determined in vivo, or in vitro, according tostandard techniques. Such activities can be a direct activity, such asan association with or an enzymatic activity on a second protein, or anindirect activity, such as a cellular signaling activity mediated byinteraction of the protein with a second protein.

For TANGO 228, biological activities include, e.g., (1) the ability tomodulate, e.g., stabilize, protein-protein interactions (e.g.,homophilic and/or heterophilic), and protein-ligand interactions, e.g.,in receptor-ligand recognition; (2) ability to interact with (e.g.,noncovalently bind to) antigens, e.g., the antigens that elicited theirformation; (3) the ability to initiate the immune response; and (4) theability to modulate the activity (e.g., the development and/oractivation) of connective tissue cells (e.g., mast cells and/ormonocytes).

Other activities of TANGO 228 include: (1) the ability to modulateintracellular signaling cascades (e.g., signal transduction cascade),e.g., by interacting with target peptides (e.g.,phosphotyrosine-containing target peptides); and (2) the ability tomediate (e.g., initiate) the allergic response, e.g., by serving as areceptor to antigens and/or a signaling molecule to other immuneresponse mediators (e.g., histamines).

Still other activities of TANGO 228 include: (1) the ability to modulate(e.g., inhibit) lipid associated processes (e.g., exocytosis and/orlipid mediator generation) by, e.g., interacting with (e.g., binding to)a cell surface protein (e.g., a receptor) on a cell type involved in theimmune response (e.g., mast cell); and (2) the ability to perform one ormore of the functions of rat surface protein MCA-32 described, forexample, in Pirozzi et al. (1995) Journal of Immunology. 155:5811-5818,the contents of which are incorporated herein by reference.

For TANGO 240, biological activities include, e.g., (1) the ability tomodulate the tuberculosis pathology pathway in the same fashion asMycobacterium tuberculosis conserved hypothetical protein Rv0712; and(2) the ability to modulate the function, migration, proliferation(e.g., suppress cell growth), and/or differentiation of cells, e.g.,cells in tissues in which it is expressed (see description of expressiondata below).

For TANGO 243, biological activities include, e.g., (1) the ability tomodulate (e.g., activate) the activity of enzymes (e.g., phospholipases)that hydrolyze lipids (e.g., phospholipids); (2) the ability to modulate(e.g., activate) the activity of enzymes that release precursors (e.g.,arachidonic acid) of regulatory molecules (e.g., prostaglandins and/oreicosanoids) associated with the arthropathy pathway; (3) the ability tomediate (e.g., activate) the arthropathy pathway, e.g., throughbiological activity (1); and (4) the ability to modulate (e.g., induce)the activity (e.g., proliferation) of cell types associated with thearthropathic pathway (e.g., leukocytes, e.g., polymorphonuclearleukocytes, and/or mononuclear inflammatory cells).

Other activities of TANGO 243 include: (1) the ability to modulate,e.g., by causing cells to alter (e.g., increase) the synthesis of,and/or by causing cells (e.g., macrophages) to release, cell mediatingmolecules, e.g., cytokines (e.g., IL-1 and/or TNF); (2) the ability tomodulate (e.g., initiate) an immune and/or inflammatory response, e.g.,through biological activity (1); and (3) the ability to modulate (e.g.,perpetuate) an immune and/or inflammatory response, e.g., throughbiological activity (1), whereby TANGO 243 itself is stimulated by thecell mediating molecule that it modulates (e.g., cytokine (e.g., IL-1and/or TNF)).

Additional activities of TANGO 243 include: (1) the ability to modulate(e.g., initiate) the activity of enzymes (e.g., phospholipases (e.g.,phospholipase A2), e.g., by modulating their signal transduction; (2)the ability to modulate (e.g., promote) cell-cell interaction (e.g.,chemotaxis) by modulating (e.g., initiating) phospholipase A2 activationand/or signal transduction; (3) the ability to modulate (e.g., increase)cell permeability (e.g., endothelial cell permeability), e.g., viabiological activity (2); and (4) the ability to modulate (e.g., promote)cell-cell adhesion (e.g., membrane fusion), e.g., via biologicalactivity (2).

Still other activities of TANGO 243 include: (1) the ability to modulate(e.g., inhibit) cellular functions, e.g., cell division, cell-fatedetermination, gene transcription, transmembrane signaling, mRNAmodification, and/or vesicle fusion; (2) the ability to modulate signaltransduction, e.g., that of transmembrane receptors; and (3) the abilityto perform one or more of the functions of human PLAP described, forexample, in Bomalaski et al. (1990) J Lab Clin Med. 16 (6):814-825, thecontents of which are incorporated herein by reference.

In one embodiment, a polypeptide of the invention has an amino acidsequence sufficiently identical to an identified domain of a polypeptideof the invention. As used herein, the term “sufficiently identical”refers to a first amino acid or nucleotide sequence which contains asufficient or minimum number of identical or equivalent (e.g., with asimilar side chain) amino acid residues or nucleotides to a second aminoacid or nucleotide sequence such that the first and second amino acid ornucleotide sequences have a common structural domain and/or commonfunctional activity. For example, amino acid or nucleotide sequenceswhich contain a common structural domain having about 60% identity,preferably 65% identity, more preferably 75%, 85%, 95%, 98% or moreidentity are defined herein as sufficiently identical.

In one embodiment, a TANGO 228 protein includes at least one or more ofthe following domains: a signal sequence, an extracellular domain, an Igdomain, a transmembrane domain, and an intracellular domain. In anotherembodiment, a TANGO 228 protein includes an extracellular domain, two Igdomains, and is wholly secreted. In yet another, a TANGO 228 proteinincludes an extracellular domain, two Ig domains, a transmembranedomain, and a cytoplasmic domain, and is a transmembrane protein.

In one embodiment, a TANGO 240 protein includes a signal peptide.

In one embodiment, a TANGO 243 protein includes a G-beta domain. Inanother embodiment, a TANGO 243 protein includes at least about twoG-beta domains. In still another embodiment, a TANGO 243 proteinincludes at least three, four, five, six, or seven G-beta domains.

The polypeptides of the present invention, or biologically activeportions thereof, can be operably linked to a heterologous amino acidsequence to form fusion proteins. The invention further featuresantibodies that specifically bind a polypeptide of the invention such asmonoclonal or polyclonal antibodies. In addition, the polypeptides ofthe invention or biologically active portions thereof can beincorporated into pharmaceutical compositions, which optionally includepharmaceutically acceptable carriers.

In another aspect, the present invention provides methods for detectingthe presence of the activity or expression of a polypeptide of theinvention in a biological sample by contacting the biological samplewith an agent capable of detecting an indicator of activity such thatthe presence of activity is detected in the biological sample.

In another aspect, the invention provides methods for modulatingactivity of a polypeptide of the invention comprising contacting a cellwith an agent that modulates (inhibits or stimulates) the activity orexpression of a polypeptide of the invention such that activity orexpression in the cell is modulated. In one embodiment, the agent is anantibody that specifically binds to a polypeptide of the invention.

In another embodiment, the agent modulates expression of a polypeptideof the invention by modulating transcription, splicing, or translationof an mRNA encoding a polypeptide of the invention. In yet anotherembodiment, the agent is a nucleic acid molecule having a nucleotidesequence that is antisense to the coding strand of an mRNA encoding apolypeptide of the invention.

The present invention also provides methods to treat a subject having adisorder characterized by aberrant activity of a polypeptide of theinvention or aberrant expression of a nucleic acid of the invention byadministering an agent which is a modulator of the activity of apolypeptide of the invention or a modulator of the expression of anucleic acid of the invention to the subject. In one embodiment, themodulator is a protein of the invention. In another embodiment, themodulator is a nucleic acid of the invention. In other embodiments, themodulator is a peptide, peptidomimetic, or other small molecule.

The present invention also provides diagnostic assays for identifyingthe presence or absence of a genetic lesion or mutation characterized byat least one of: (i) aberrant modification or mutation of a geneencoding a polypeptide of the invention, (ii) mis-regulation of a geneencoding a polypeptide of the invention, and (iii) aberrantpost-translational modification of the invention wherein a wild-typeform of the gene encodes a protein having the activity of thepolypeptide of the invention.

In another aspect, the invention provides a method for identifying acompound that binds to or modulates the activity of a polypeptide of theinvention. In general, such methods entail measuring a biologicalactivity of the polypeptide in the presence and absence of a testcompound and identifying those compounds which alter the activity of thepolypeptide.

The invention also features methods for identifying a compound whichmodulates the expression of a polypeptide or nucleic acid of theinvention by measuring the expression of the polypeptide or nucleic acidin the presence and absence of the compound.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C depict the cDNA sequence of human TANGO 228 (SEQ ID NO:1)and the predicted amino acid sequence of TANGO 228 (SEQ ID NO:3). Theopen reading frame of SEQ ID NO:1 extends from nucleotide 34 tonucleotide 1062 of SEQ ID NO:1 (SEQ ID NO:2).

FIG. 2 depicts a hydropathy plot of human TANGO 228. Relativelyhydrophobic regions of the protein are above the dashed horizontal line,and relatively hydrophilic regions of the protein are below the dashedhorizontal line. The cysteine residues (cys) and potentialN-glycosylation sites (Ngly) are indicated by short vertical lines justbelow the hydropathy trace. The dashed vertical line separates thesignal sequence (amino acids 1 to 19 of SEQ ID NO:3; SEQ ID NO:5) on theleft from the mature protein (amino acids 20 to 343 of SEQ ID NO:3; SEQID NO:4) on the right. Thicker gray horizontal bars below the dashedhorizontal line indicate extracellular (“out”), transmembrane (“TM”),and intracellular (“in”) regions of the molecule. Below the hydropathyplot, the amino acid sequence of TANGO 228 is depicted.

FIGS. 3A-3H depict an alignment of the nucleotide sequence of ratsurface protein MCA-32 (SEQ ID NO:11; GenBank Accession Number U39546)and the nucleotide sequence of human TANGO 228 (SEQ ID NO:1). Thenucleotide sequences of rat MCA-32 and human TANGO 228 are 28.3%identical. This alignment was performed using the ALIGN alignmentprogram with a PAM120 scoring matrix, a gap length penalty of 12, and agap penalty of 4.

FIGS. 4A-4C depict an alignment of the nucleotide sequence of the openreading frames of rat MCA-32 (nucleotides 8 to 826 of SEQ ID NO:11) andhuman TANGO 228 (SEQ ID NO:2). The nucleotide sequences of the openreading frames of rat MCA-32 and human TANGO 228 (SEQ ID NO:2) are 45.4%identical. This alignment was performed using the ALIGN alignmentprogram with a PAM120 scoring matrix, a gap length penalty of 12, and agap penalty of 4.

FIG. 5 depicts an alignment of the amino acid sequence of rat MCA-32(SEQ ID NO:12) and the amino acid sequence of human TANGO 228 (SEQ IDNO:3). The amino acid sequences of rat MCA-32 and human TANGO 228 are26.8% identical. This alignment was performed using the ALIGN alignmentprogram with a PAM120 scoring matrix, a gap length penalty of 12, and agap penalty of 4.

FIGS. 6A-6B depict the cDNA sequence of human TANGO 240 (SEQ ID NO:13)and the predicted amino acid sequence of TANGO 240 (SEQ ID NO:15). Theopen reading frame of SEQ ID NO:13 extends from nucleotide 2 tonucleotide 1123 of SEQ ID NO:13 (SEQ ID NO:14).

FIG. 7 depicts a hydropathy plot of human TANGO 240. Relativelyhydrophobic regions of the protein are shown above the horizontal line,and relatively hydrophilic regions of the protein are below thehorizontal line. The cysteine residues (cys) and potentialN-glycosylation sites (Ngly) are indicated by short vertical lines justbelow the hydropathy trace. The dashed vertical line separates thesignal sequence (amino acids 1 to 34 of SEQ ID NO:15; SEQ ID NO:17) onthe left from the mature protein (amino acids 35 to 374 of SEQ ID NO:15;SEQ ID NO:16) on the right. Below the hydropathy plot, the amino acidsequence of TANGO 240 is depicted.

FIG. 8 depicts an alignment of the amino acid sequence of theMycobacterium tuberculosis conserved hypothetical protein Rv0712 (SEQ IDNO:18; GenBank Accession Number Z84395) and the amino acid sequence ofhuman TANGO 240 (SEQ ID NO:15). The amino acid sequences of Rv0712 andhuman TANGO 1240 are 31.2% identical. This alignment was performed usingthe ALIGN alignment program with a PAM120 scoring matrix, a gap lengthpenalty of 12, and a gap penalty of 4.

FIGS. 9A-9C depict the cDNA sequence of human TANGO 243 (SEQ ID NO:19)and the predicted amino acid sequence of TANGO 243 (SEQ ID NO:21). Theopen reading frame of SEQ ID NO:19 extends from nucleotide 183 tonucleotide 2567 of SEQ ID NO:19 (SEQ ID NO:20).

FIG. 10 depicts a hydropathy plot of human TANGO 243. Relativelyhydrophobic regions of the protein are above the dashed horizontal line,and relatively hydrophilic regions of the protein are below the dashedhorizontal line. The cysteine residues (cys) and potentialN-glycosylation sites (Ngly) are indicated by short vertical lines justbelow the hydropathy trace. Below the hydropathy plot, the amino acidsequence of TANGO 243 is depicted.

FIGS. 11A-11F depict an alignment of the nucleotide sequence of humanPLAP (SEQ ID NO:29; GenBank Accession Number AF083395) and thenucleotide sequence of human TANGO 243 (SEQ ID NO:19). The nucleotidesequences of human PLAP and human TANGO 243 are 78.8% identical. Thisalignment was performed using the ALIGN alignment program with a PAM120scoring matrix, a gap length penalty of 12, and a gap penalty of 4.

FIGS. 12A-12E depict an alignment of the nucleotide sequence of the openreading frames of human PLAP (nucleotides 1 to 2217 of SEQ ID NO:29) andhuman TANGO 243 (SEQ ID NO:20). The nucleotide sequences of the openreading frames of human PLAP and human TANGO 243 (SEQ ID NO:20) are92.7% identical. This alignment was performed using the ALIGN alignmentprogram with a PAM120 scoring matrix, a gap length penalty of 12, and agap penalty of 4.

FIGS. 13A-13B depict an alignment of the amino acid sequence of humanPLAP (SEQ ID NO:30) and the amino acid sequence of human TANGO 243 (SEQID NO:21). The amino acid sequences of human PLAP and human TANGO 243are 92.8% identical. This alignment was performed using the ALIGNalignment program with a PAM120 scoring matrix, a gap length penalty of12, and a gap penalty of 4.

FIG. 14 depicts the cDNA sequence of murine TANGO 228 (SEQ ID NO:31) andthe predicted amino acid sequence of murine TANGO 228 (SEQ ID NO:33).The open reading frame of SEQ ID NO:31 extends from nucleotide 27 tonucleotide 743 of SEQ ID NO:31 (SEQ ID NO:33).

FIGS. 15A-15B depict an alignment of the nucleotide sequence of murineTANGO 228 (SEQ ID NO:31; upper line of each pair) and the nucleotidesequence of human TANGO 228 (SEQ ID NO:1; lower line of each pair). Thenucleotide sequences of murine TANGO 228 and human TANGO 228 are 58.2%identical, over the length of the murien cDNA. This alignment wasperformed using the ALIGN alignment program with a PAM120 scoringmatrix, a gap length penalty of 12, and a gap penalty of 4.

FIGS. 16A-16B depict an alignment of the nucleotide sequence of the openreading frames of murine TANGO 228 (SEQ ID NO:33; upper line of eachpair) and human TANGO 228 (SEQ ID NO:3; lower line of each pair). Thenucleotide sequences of the open reading frames of murine TANGO 228human TANGO 228 are 58.2% identical, over the length of the murine ORF.This alignment was performed using the ALIGN alignment program with aPAM120 scoring matrix, a gap length penalty of 12, and a gap penalty of4.

FIG. 17 depicts an alignment of the amino acid sequence of murine TANGO228 (SEQ ID NO:32; upper line of each pair) and human TANGO 228 (SEQ IDNO:3; lower line of each pair). The amino acid sequences of murine TANGO228 and human TANGO 228 are 30.6% identical, over the length of themurine protein. This alignment was performed using the ALIGN alignmentprogram with a PAM120 scoring matrix, a gap length penalty of 12, and agap penalty of 4.

FIGS. 18A-18B depict the cDNA sequence of murine TANGO 240 (SEQ IDNO:39) and the predicted amino acid sequence of murine TANGO 240 (SEQ IDNO:41). The open reading frame of SEQ ID NO:41 extends from nucleotide 2to nucleotide 1117 of SEQ ID NO:41 (SEQ ID NO:40).

FIGS. 19A-19D depict an alignment of the nucleotide sequence of murineTANGO 240 (SEQ ID NO:39; upper line of each pair) and the nucleotidesequence of human TANGO 228 (SEQ ID NO:13; lower line of each pair). Inthis alignment the sequences are 78.4% identical, over the length of thehuman cDNA. This alignment was performed using the ALIGN alignmentprogram with a PAM120 scoring matrix, a gap length penalty of 12, and agap penalty of 4.

FIGS. 20A-20B depict an alignment of the nucleotide sequence of the openreading frames of murine TANGO 240 (SEQ ID NO:40; upper line of eachpair) and human TANGO 240 (SEQ ID NO:14; lower line of each pair). Thenucleotide sequences of the open reading frames of murine TANGO 240human TANGO 240 are 84.4% identical. This alignment was performed usingthe ALIGN alignment program with a PAM120 scoring matrix, a gap lengthpenalty of 12, and a gap penalty of 4.

FIG. 21 depicts an alignment of the amino acid sequence of murine TANGO240 (SEQ ID NO:41; upper line of each pair) and human TANGO 240 (SEQ IDNO:17; lower line of each pair). The amino acid sequences of murineTANGO 240 and human TANGO 240 are 86% identical. This alignment wasperformed using the ALIGN alignment program with a PAM120 scoringmatrix, a gap length penalty of 12, and a gap penalty of 4.

FIG. 22A-22C depict the cDNA sequence of human TANGO 243 (SEQ ID NO:19)and the predicted amino acid sequence of a longer form of TANGO 243 (SEQID NO:45) arising from an alternative translation of the human TANGO 243cDNA. In this translation the open reading frame of SEQ ID NO:19 extendsfrom nucleotide 3 to nucleotide 2567 of SEQ ID NO:19 (SEQ ID NO:44).

FIG. 23 depicts a hydropathy plot of the longer form of human TANGO 243arising from the alternative translation of the human TANGO 243 cDNA.Relatively hydrophobic regions of the protein are above the dashedhorizontal line, and relatively hydrophilic regions of the protein arebelow the dashed horizontal line. The cysteine residues (cys) andpotential N-glycosylation sites (Ngly) are indicated by short verticallines just below the hydropathy trace. Below the hydropathy plot, theamino acid sequence of TANGO 243 is depicted.

DETAILED DESCRIPTION OF THE INVENTION

The TANGO 228, TANGO 240, and TANGO 243 proteins and nucleic acidmolecules comprise a family of molecules having certain conservedstructural and functional features. As used herein, the term “family” isintended to mean two or more proteins or nucleic acid molecules having acommon structural domain and having sufficient amino acid or nucleotidesequence identity as defined herein. Family members can be from eitherthe same or different species. For example, a family can comprises twoor more proteins of human origin, or can comprise one or more proteinsof human origin and one or more of non-human origin. Members of the samefamily may also have common structural domains.

For example, TANGO 228 proteins and TANGO 240 proteins of the inventionhave signal sequences. As used herein, a “signal sequence” includes apeptide of at least about 15 or 20 amino acid residues in length whichoccurs at the N-terminus of secretory and membrane-bound proteins andwhich contains at least about 70% hydrophobic amino acid residues suchas alanine, leucine, isoleucine, phenylalanine, proline, tyrosine,tryptophan, or valine. In a preferred embodiment, a signal sequencecontains at least about 10 to 40 amino acid residues, preferably about19-34 amino acid residues, and has at least about 60-80%, morepreferably 65-75%, and more preferably at least about 70% hydrophobicresidues. A signal sequence serves to direct a protein containing such asequence to a lipid bilayer. Thus, in one embodiment, a TANGO 228protein contains a signal sequence at about amino acids 1 to 19 of SEQID NO:3 (SEQ ID NO:5). In another embodiment, a TANGO 240 proteincontains a signal sequence at about amino acids 1 to 34 of SEQ ID NO:15(SEQ ID NO:17). The signal sequence is cleaved during processing of themature protein.

In another example, a TANGO 228 family member also includes one or moreof the following domains: (1) an extracellular domain; (2) atransmembrane domain; and (3) a cytoplasmic domain. Thus, in oneembodiment, a TANGO 228 protein contains an extracellular domain atabout amino acids 1 to 227 of SEQ ID NO:3 (SEQ ID NO:6). In anotherembodiment, a TANGO 228 protein contains a transmembrane domain at aboutamino acids 228 to 249 of SEQ ID NO:3 (SEQ ID NO:7). In anotherembodiment, a TANGO 228 protein contains a cytoplasmic domain at aboutamino acids 250 to 343 of SEQ ID NO:3 (SEQ ID NO:10).

In one embodiment, the extracellular domain of TANGO 228 can alsoinclude an Ig domain. In another embodiment, the extracellular domain ofTANGO 228 includes about 1 to 10, preferably about 3-8, more preferablyabout 6, N-glycosylation sites, about 1 to 30, preferably about 10 to20, more preferably about 15 conserved serine residues (not includingresidues within the Ig domain), and about 1 to 20, preferably about 5 to15, more preferably about 11 conserved threonine residues (not includingresidues within the Ig domain).

In a preferred embodiment, a TANGO 228 family member has the amino acidsequence of SEQ ID NO:3 wherein the extracellular domain is located atabout amino acids 1 to 227 (SEQ ID NO:6), the N-glycosylation sites arelocated at about amino acid residue positions 51 to 54, 60 to 63, 89 to92, 151 to 154, 157 to 160, and 182 to 185, the conserved serineresidues are located at about amino acid positions 3, 6, 12, 15, 16, 36,41, 109, 111, 114, 118, 127, 209, 216, and 221, and the conservedthreonine residues are located at about amino acid positions 18, 31, 43,108, 120, 126, 137, 139, 207, 213, and 217.

TANGO 228 family members can also include an Ig domain contained withinthe extracellular domain. As used herein, the term “Ig domain” refers toa protein domain bearing homology to immunoglobulin superfamily members.An Ig domain includes about 30-90 amino acid residues, preferably about40-80 amino acid residues, more preferably about 50-70 amino acidresidues, still more preferably about 55-65 amino acid residues, andmost preferably about 57 to 59 amino acid residues. Typically, an Igdomain contains a conserved cysteine residue within about 5 to 15 aminoacid residues, preferably about 7 to 12 amino acid residues, and mostpreferably about 8 amino acid residues from its N-terminal end, andanother conserved cysteine residue within about 1 to 5 amino acidresidues, preferably about 2 to 4 amino acid residues, and mostpreferably about 3 amino acid residues from its C-terminal end.

An Ig domain typically has the following consensus sequence, beginningabout 1 to 15 amino acid residues, more preferably about 3 to 10 aminoacid residues, and most preferably about 5 amino acid residues from thedomain C terminal end of a protein: (FY)-Xaa-C-Xaa-(VA)-COO—, wherein(FY) is either a phenylalanine or a tyrosine residue (preferablytyrosine), Xaa is any amino acid, C is a cysteine residue, (VA) iseither a valine or an alanine residue (preferably alanine), and COO— isthe protein C terminus.

In one embodiment, a TANGO 228 family member includes one or more Igdomains having an amino acid sequence that is at least about 55%,preferably at least about 65%, more preferably at least about 75%, yetmore preferably at least about 85%, and most preferably at least about95% identical to amino acids 49 to 105 and/or amino acids 140 to 198 ofSEQ ID NO:3, which are the Ig domains of TANGO 228 (these Ig domains arealso represented as SEQ ID NO:8 and 9, respectively). In anotherembodiment, a TANGO 228 family member includes one or more Ig domainshaving an amino acid sequence that is at least about 55%, preferably atleast about 65%, more preferably at least about 75%, yet more preferablyat least about 85%, and most preferably at least about 95% identical toamino acids 49 to 105 and/or amino acids 140 to 198 of SEQ ID NO:3 (SEQID NO:8 and 9, respectively), includes a conserved cysteine residueabout 8 residues downstream from the N-terminus of the Ig domain, andhas one or more Ig domain consensus sequences described herein. Inanother embodiment, a TANGO 228 family member includes one or more Igdomains having an amino acid sequence that is at least 55%, preferablyat least about 65%, more preferably at least about 75%, yet morepreferably at least about 85%, and most preferably at least about 95%identical to amino acids 49 to 105 and/or amino acids 140 to 198 of SEQID NO:3 (SEQ ID NO:8 and 9, respectively), includes a conserved cysteineresidue 8 residues downstream from the N-terminus of the Ig domain, hasone or more Ig domain consensus sequences described herein, and has aconserved cysteine within the consensus sequence that forms a disulfideboth with said first conserved cysteine. In yet another embodiment, aTANGO 228 family member includes one or more Ig domains having an aminoacid sequence that is at least 55%, preferably at least about 65%, morepreferably at least about 75%, yet more preferably at least about 85%,and most preferably at least about 95% identical to amino acids 49 to105 and/or amino acids 140 to 198 of SEQ ID NO:3 (SEQ ID NO:8 and 9,respectively), includes a conserved cysteine residue 8 residuesdownstream from the N-terminus of the Ig domain, has one or more Igdomain consensus sequences described herein, has a conserved cysteinewithin the consensus sequence that forms a disulfide both with saidfirst conserved cysteine, and has at least one TANGO 228 biologicalactivity as described herein.

In a preferred embodiment, a TANGO 228 family member has the amino acidsequence of SEQ ID NO:3 wherein the aforementioned Ig domain conservedresidues are located as follows: the N-terminal conserved cysteineresidue is located at about amino acid residue position 56 (within thefirst Ig domain (SEQ ID NO:8)) and at about amino acid residue position147 (within the second Ig domain (SEQ ID NO:9)), and the C-terminalconserved cysteine residue is located at about amino acid position 103(within the first Ig domain (SEQ ID NO:8)) and at about amino acidresidue position 196 (within the second Ig domain (SEQ ID NO:9)).

A TANGO 228 family member can also include a cytoplasmic domain. Thecytoplasmic domain can include hydrophobic amino acid residues, and cancontain several SH2 domain recognition sites, which contain typicallybegin with a tyrosine residue. This cytoplasmic domain can contain about1 to 5, more preferably about 2 to 4, and still more preferably about 3SH2 domain recognition sites.

One type of SH2 domain recognition site typically has the followingconsensus sequence: Y-Xaa1-Xaa1-(IP), wherein Y is a tyrosine residue,Xaa1 is any hydrophobic amino acid, and (IP) is either an isoleucine orproline residue. Another type of SH2 domain recognition site typicallyhas the following consensus sequence: Y-Xaa1-Xaa-Xaa1, wherein Y is atyrosine residue, Xaa1 is any hydrophobic amino acid, and Xaa is anyamino acid.

In a preferred embodiment, a TANGO 228 family member has the amino acidsequence of SEQ ID NO:3 wherein the cytoplasmic domain is located atabout amino acids 250 to 343 (SEQ ID NO:10) and the conserved SH2 domainrecognition sites are located at about amino acid positions 276 to 279,313 to 317, and 338 to 341.

A TANGO 240 family member can include a signal sequence. In a preferredembodiment, a TANGO 240 family member has the amino acid sequence of SEQID NO:15, and the signal sequence is located at about amino acids 1 to34.

A TANGO 243 family member can include one or more G-beta domains. Asused herein, a “G-beta domain” refers to a domain which includes about25 to 55 amino acid residues, preferably about 30 to 50 amino acidresidues, more preferably about 35-55 amino acid residues, and mostpreferably about 38 to 44 amino acid residues. In addition, a G-betadomain contains a conserved glycine residue adjacent to a conservedhistidine residue, a conserved aspartic acid residue, and one or moreconserved valine or isoleucine residues.

G-beta domains are typically found within the WD-repeat(tryptophan-aspartate repeat) family of proteins. The WD-repeat familyof proteins are found in all eukaryotes but not in prokaryotes, and arefunctionally diverse, in some cases regulating cellular functions,transmembrane signaling, and/or vesicle fusion, among other things.

A G-beta domain typically has the following consensus sequence:G-H-Xaa(n1)-V-Xaa(n2)-[V or L]-Xaa(n3)-D-Xaa(n4)-W, wherein G isglycine, H is histidine, V is valine, D is aspartic acid, L is leucine,W is tryptophan, Xaa is any amino acid, n1 is about 1-8 amino acidresidues, more preferably about 2-5 amino acid residues, and morepreferably about 3 amino acid residues, n2 is about 1-8 amino acidresidues, more preferably about 1-4 amino acid residues, and morepreferably about 2 amino acid residues, n3 is about 5-30 amino acidresidues, more preferably about 10-25 amino acid residues, and morepreferably about 14-19 amino acid residues, and n4 is about 1-8 aminoacid residues, more preferably about 3-6 amino acid residues, and morepreferably about 5 amino acid residues.

A TANGO 243 family member can also include a G-beta-like domain. As usedherein, a “G-beta-like domain” refers to an stretch of amino acidresidues, about 25 to 55 residues, preferably about 30 to 50 residues,more preferably about 35-55 amino acid residues, and most preferablyabout 38 to 44 residues, in length, that is similar in content to aG-beta domain. A G-beta-like domain typically has the followingconsensus sequence: [V or I]-Xaa(2)-[V or C]-Xaa(n1)-[P or D]-[D orN]-G-Xaa(n2)-G-Xaa(2)-D, wherein V is valine, I is isoleucine, C iscysteine, P is proline, D is aspartic acid, G is glycine, Xaa is anyamino acid, n1 is about 1 to 8 amino acid residues, preferably about 2to 5 amino acid residues, more preferably about 3 amino acid residues,and n2 is about 1 to 8 amino acid residues, preferably about 2 to 5amino acid residues, more preferably about 4 amino acid residues.

In one embodiment, a TANGO 243 family member includes one or more G-betadomains having an amino acid sequence that is at least about 55%,preferably at least about 65%, more preferably at least about 75%, yetmore preferably at least about 85%, and most preferably at least about95% identical to amino acids 8 to 47, or amino acids 55 to 98, or aminoacids 102 to 139, or amino acids 141 to 179, or amino acids 181 to 218,or amino acids 221 to 259 of SEQ ID NO:21, which are the G-beta domainsof TANGO 243 (these G-beta domains are also represented as SEQ ID NO:22,23, 24, 25, 26, and 27, respectively). In another embodiment, a TANGO243 family member includes one or more G-beta domains having an aminoacid sequence that is at least about 55%, preferably at least about 65%,more preferably at least about 75%, yet more preferably at least about85%, and most preferably at least about 95% identical to amino acids 8to 47, or amino acids 55 to 98, or amino acids 102 to 139, or aminoacids 141 to 179, or amino acids 181 to 218, or amino acids 221 to 259of SEQ ID NO:21 (SEQ ID NO:22, 23, 24, 25, 26, and 27, respectively),and has a G-beta domain consensus sequence as described herein. In yetanother embodiment, a TANGO 243 family member includes one or moreG-beta domains having an amino acid sequence that is at least about 55%,preferably at least about 65%, more preferably at least about 75%, yetmore preferably at least about 85%, and most preferably at least about95% identical to amino acids 8 to 47, or amino acids 55 to 98, or aminoacids 102 to 139, or amino acids 141 to 179, or amino acids 181 to 218,or amino acids 221 to 259 of SEQ ID NO:21 (SEQ ID NO:22, 23, 24, 25, 26,and 27, respectively), has a G-beta domain consensus sequence asdescribed herein, and has at least one TANGO 243 biological activity asdescribed herein.

In another embodiment, a TANGO 243 family member includes one or moreG-beta domains having an amino acid sequence that is at least about 55%,preferably at least about 65%, more preferably at least about 75%, yetmore preferably at least about 85%, and most preferably at least about95% identical to amino acids 8 to 47, or amino acids 55 to 98, or aminoacids 102 to 139, or amino acids 141 to 179, or amino acids 181 to 218,or amino acids 221 to 259 of SEQ ID NO:21, amino acids 68 to 107, oramino acids 115 to 158, or amino acids 162 to 199, or amino acids 201 to139, or amino acids 141 to 278, or amino acids 281 to 219 of SEQ IDNO:45 (SEQ ID NO:22, 23, 24, 25, 26, and 27, respectively), G-beta-likedomain having an amino acid sequence that is at least about 55%,preferably at least about 65%, more preferably at least about 75%, yetmore preferably at least about 85%, and most preferably at least about95% identical to amino acids 261 to 298 of SEQ ID NO:21 (SEQ ID NO:28),has a G-beta domain consensus sequence and a G-beta-like domainconsensus sequence as described herein, and has at least one TANGO 243biological activity as described herein.

A TANGO 243 family member can include a PLAP-like domain. A PLAP-likedomain 1 can include at least 20, 25, 30, or 40 contiguous amino acidsof SEQ ID NO:47. A PLAP-like domain 2 can include at least 20, 25, 30,40, or 45 contiguous amino acids of SEQ ID NO:48. A PLAP-like domain 3can include 20, 25, 30, 40, 45, 50, 60, 70, or 80 contiguous amino acidsof SEQ ID NO:49. A PLAP-like domain 1 can include at least 20, 25, 30,or 35 contiguous amino acids of SEQ ID NO:50.

In a preferred embodiment, a TANGO 243 family member has the amino acidsequence of SEQ ID NO:21 wherein the G-beta consensus sequences arelocated from amino acid 16 to 46 (the first G-beta domain (SEQ IDNO:22)), 63 to 97 (the second G-beta domain (SEQ ID NO:23)), 110 to 138(the third G-beta domain (SEQ ID NO:24)), 149 to 178 (the fourth G-betadomain (SEQ ID NO:25)), 189 to 217 (the fifth G-beta domain (SEQ IDNO:26)), and 229 to 258 (the sixth G-beta domain (SEQ ID NO:27)), andthe G-beta-like consensus sequence is located from amino acid 261 to 298(SEQ ID NO:28).

Various features of human TANGO 228, murine TANGO 228, human TANGO 240,and human TANGO 243 are summarized below.

Human TANGO 228

A cDNA encoding human TANGO 228 was identified by analyzing thesequences of clones present in a fetal spleen cDNA library. Thisanalysis led to the identification of a clone, jthsa055f08, encodingfull-length human TANGO 228. The human TANGO 228 cDNA of this clone is4043 nucleotides long (FIGS. 1A-1C; SEQ ID NO:1). The open reading frameof this cDNA, nucleotides 34 to 1062 of SEQ ID NO:1 (SEQ ID NO:2),encodes a 343 amino acid transmembrane protein (FIGS. 1A-1C; SEQ IDNO:3).

The signal peptide prediction program SIGNALP (Nielsen et al. (1997)Protein Engineering 10:1-6) predicted that human TANGO 228 includes a 19amino acid signal peptide (amino acid 1 to about amino acid 19 of SEQ IDNO:3) (SEQ ID NO:5) preceding the mature TANGO 228 protein(corresponding to about amino acid 20 to amino acid 343 of SEQ ID NO:3)(SEQ ID NO:4). The TANGO 228 protein molecular weight is 38.7 kDa priorto the cleavage of the signal peptide, 36.4 kDa after cleavage of thesignal peptide.

TANGO 228 includes a extracellular domain (about amino acids 1 to 227 ofSEQ ID NO:3; SEQ ID NO:6), a transmembrane domain (about amino acids 228to 249 of SEQ ID NO:3; SEQ ID NO:7), two Ig domains (about amino acids49 to 105, and about amino acids 140 to 198, of SEQ ID NO:3; SEQ ID NO:8and 9), and a cytoplasmic domain (about amino acids 250 to 343 of SEQ IDNO:3; SEQ ID NO:10).

An N-glycosylation site having the sequence NVSM is found from aminoacids 51 to 54 of SEQ ID NO:3. A second N-glycosylation site having thesequence NKSL is found from amino acids 60 to 63. A thirdN-glycosylation site having the sequence NLSI is found from amino acids89 to 92. A fourth N-glycosylation site having the sequence NGSL isfound from amino acids 151 to 154. A fifth N-glycosylation site havingthe sequence NYTF is found from amino acids 157 to 160. A sixthN-glycosylation site having the sequence NLTK is found from amino acids182 to 185. A cAMP and cGMP-dependent protein kinase phosphorylationsite having the sequence RRKT is found from amino acids 71 to 74. Aprotein kinase C phosphorylation site having the sequence TCR is foundfrom amino acids 18 to 20. A second protein kinase C phosphorylationsite having the sequence SHK is found from amino acids 57 to 59. A thirdprotein kinase C phosphorylation site having the sequence TDR is foundfrom amino acids 139 to 141. A fourth protein kinase C phosphorylationsite having the sequence TKK is found from amino acids 184 to 186. Afifth protein kinase C phosphorylation site having the sequence TRK isfound from amino acids 254 to 256. A sixth protein kinase Cphosphorylation site having the sequence SYK is found from amino acids331 to 333. A casein kinase II phosphorylation site having the sequenceSITE is found from amino acids 91 to 94. A second casein kinase IIphosphorylation site having the sequence TIVD is found from amino acids120 to 123. A third casein kinase II phosphorylation site having thesequence TETD is found from amino acids 137 to 140. A fourth caseinkinase II phosphorylation site having the sequence TFFE is found fromamino acids 159 to 162. A fifth casein kinase II phosphorylation sitehaving the sequence SKYD is found from amino acids 172 to 175. A sixthcasein kinase II phosphorylation site having the sequence TGGD is foundfrom amino acids 217 to 220. A seventh casein kinase II phosphorylationsite having the sequence TAME is found from amino acids 269 to 272. Aneighth casein kinase II phosphorylation site having the sequence SVPE isfound from amino acids 288 to 291. A ninth casein kinase IIphosphorylation site having the sequence TAQD is found from amino acids300 to 303. A tyrosine kinase phosphorylation site having the sequenceKNPGEEEEY is found from amino acids 186 to 194. A second tyrosine kinasephosphorylation site having the sequence KHSQELQY is found from aminoacids 306 to 313. An N-myristoylation site having the sequence GQNVSM isfound from amino acids 49 to 54. A second N-myristoylation site havingthe sequence GTQDGK is found from amino acids 77 to 82. A thirdN-myristoylation site having the sequence GIYANI is found from aminoacids 274 to 279. A fourth N-myristoylation site having the sequenceGSRPCV is found from amino acids 293 to 298. A cell attachment sequencehaving the sequence RGD is found from amino acids 266 to 268. A leucinezipper pattern having the sequence LLLPGLLLLLVVIILILAFWVL is found fromamino acids 228 to 249.

TANGO 228 is homologous to rat MCA-32, which is known to exist in twoforms, one of which has a transmembrane domain, and the other which doesnot. Thus TANGO 228, like rat MCA-32, is likely to exist in two forms,one having domains as follows and is wholly secreted: an extracellulardomain (SEQ ID NO:6) containing one or more Ig domains (SEQ ID NO:8 and9), and the other having the following domains and is a transmembrane,e.g., a cell surface, protein: an extracellular domain (SEQ ID NO:6)containing one or more Ig domains (SEQ ID NO:8 and 9), a transmembranedomain (SEQ ID NO:7), and cytoplasmic domain (SEQ ID NO:10).

FIGS. 3A-3H show an alignment of the human TANGO 228 full length nucleicacid sequence (SEQ ID NO:1) with the rat MCA-32 full length nucleic acidsequence (SEQ ID NO:11). FIGS. 4A-4C show an alignment of the humanTANGO 228 coding region (SEQ ID NO:2) with the rat MCA-32 coding region.FIG. 5 shows an alignment of the human TANGO 228 protein sequence (SEQID NO:3) with the rat MCA-32 protein sequence (SEQ ID NO:12). As shownin FIG. 5, the human TANGO 228 signal sequence is represented by aminoacids 1-19 (and encoded by nucleotides 34 to 90 of SEQ ID NO:1). Thoughthe rat MCA-32 gene structure suggests it can function as a secretedprotein or cell surface protein, depending on the splice variant, itdoes not exhibit the characteristic hydrophobic signal sequencegenerally found in secretory and transmembrane proteins. The human TANGO228 extracellular domain sequence (SEQ ID NO:6) is represented by aminoacids 1 to 227 (and encoded by nucleotides 34 to 714 of SEQ ID NO:1),and the rat MCA-32 extracellular domain sequence is represented by aminoacids 1 to 177 (and encoded by nucleotides 8 to 523 of SEQ ID NO:11).The human TANGO 2281 g domains (SEQ ID NO:8 and 9) are represented byamino acids 49 to 105, and 140 to 198 (and encoded by nucleotides 178 to348, and 451 to 627 of SEQ ID NO:1), and the rat MCA-321 g domain isrepresented by amino acids 99 to 153 (and encoded by nucleotides 287 to451 of SEQ ID NO:11). The human TANGO 228 transmembrane domain (SEQ IDNO:7) is represented by amino acids 228 to 249 (and encoded bynucleotides 715 to 780 of SEQ ID NO:1), and the rat MCA-32 transmembranedomain is represented by amino acids 178 to 199 (and encoded bynucleotides 524 to 589 of SEQ ID NO:11). The human TANGO 228 cytoplasmicdomain (SEQ ID NO:10) is represented by amino acids 250 to 343 (andencoded by nucleotides 781 to 1065 of SEQ ID NO:1), and the rat MCA-32cytoplasmic domain is represented by amino acids 200 to 278 (and encodedby nucleotides 590 to 829 of SEQ ID NO:11).

FIGS. 3A-3H and FIGS. 4A-4C show that there is an overall 28.3% identitybetween the full length human TANGO 228 nucleic acid molecule and thefull length rat MCA-32 nucleic acid molecule, and an overall 45.5%identity between the open reading frame of human TANGO 228 nucleic acidmolecule and the open reading frame of the rat MCA-32 nucleic acidmolecule, respectively. The amino acid alignment in FIG. 5 shows a 26.8%overall amino acid sequence identity between human TANGO 228 and ratMCA-32.

Clone EpT228, which encodes human TANGO 228, was deposited with theAmerican Type Culture Collection (10801 University Boulevard, Manassas,Va. 20110-2209) on Feb. 18, 1999 and assigned Accession Number 207116.This deposit will be maintained under the terms of the Budapest Treatyon the International Recognition of the Deposit of Microorganisms forthe Purposes of Patent Procedure. This deposit was made merely as aconvenience for those of skill in the art and is not an admission that adeposit is required under 35 U.S.C. § 112.

FIG. 2 depicts a hydropathy plot of human TANGO 228. Relativelyhydrophobic regions of the protein are shown above the horizontal line,and relatively hydrophilic regions of the protein are below thehorizontal line. The cysteine residues (cys) and N-glycosylation sitesare indicated by short vertical lines just below the hydropathy trace.The dashed vertical line separates the signal sequence (amino acids 1 to19 of SEQ ID NO:3; SEQ ID NO:5) on the left from the mature protein(amino acids 20 to 343 of SEQ ID NO:3; SEQ ID NO:4) on the right. TheTANGO 228 transmembrane domain is indicated by the section of the plotunder which the number 7.1 can be seen, which represents a scoreassigned to the predicted transmembrane domain. The extracellular domain(SEQ ID NO:6) and cytoplasmic domain (SEQ ID NO:10) are similarlyindicated by gray horizontal bars, labeled as “out” and “in”,respectively.

Human TANGO 228 was mapped to h17q23. The nearby flanking markersinclude: WI-4118 and WI-5110. The known nearby loci include: JPD(periodontitis, juvenile) and DGI1 (dentinogenesis imperfecta). Nearbyknown human genes include: COIL (coilin P80), TNFAIP1 (tumor necrosisfactor), GH1 (growth hormone1, 2), ICAM2 (intercellular adhesionmolecule), APOH (apolipoprotein H), PEPE(peptidase E), MPO(myeloperoxidase), and CDK3 (cyclin dependent kinase3). Thus, humanTANGO 228 nucleic acid molecules can be used to map these loci andgenes.

The syntenic mouse chromosomal location is mo11. Nearby mouse lociinclude: rimy (rimy), al (alopecia), nog (noggin), and bda(baldarthritic). Near known mouse genes include: rimy (rimy), al(alopecia), hlf (hepatic leukemia factor), chad (ahondroadherin), re(rex), mpo (myeloperoxidase), nog (noggin), bda (bald arthritic),gip(gastric inhibitory polypeptide), and ngfr (nerve growth factorreceptor).

Murine TANGO 228

A cDNA encoding murine TANGO 228 was identified by analyzing thesequences of clones present in a bone marrow stromal cell cDNA library.This analysis led to the identification of a clone, jtmMa107f05,encoding full-length murine TANGO 228. The murine TANGO 228 cDNA of thisclone is 911 nucleotides long (FIG. 14; SEQ ID NO:31). The open readingframe of this cDNA, nucleotides 27 to 743 of SEQ ID NO:31 (SEQ IDNO:32), encodes a 239 amino acid transmembrane protein (FIG. 14; SEQ IDNO:33).

The signal peptide prediction program SIGNALP (Nielsen et al. (1997)Protein Engineering 10:1-6) predicted that murine TANGO 228 includes a34 amino acid signal peptide (amino acid 1 to about amino acid 34 of SEQID NO:33; SEQ ID NO:35) preceding the mature murine TANGO 228 protein(corresponding to about amino acid 35 to amino acid 239 of SEQ ID NO:33;SEQ ID NO:34). The murine TANGO 228 protein molecular weight is 26.7 kDaprior to the cleavage of the signal peptide, 22.9 kDa after cleavage ofthe signal peptide.

Mature murine TANGO 228 includes an extracellular domain (about aminoacids 35 to 141 of SEQ ID NO:33; SEQ ID NO:36), a transmembrane domain(about amino acids 142 to 164 of SEQ ID NO:33; SEQ ID NO:37), and acytoplasmic domain (about amino acids 165 to 239 of SEQ ID NO:33; SEQ IDNO:8). Murine TANGO 228 also includes an Ig domain (about amino acids 59to 115 of SEQ ID NO:33; SEQ ID NO:46).

FIGS. 15A-15B show an alignment of the murine TANGO 228 full lengthnucleic acid sequence (upper line of each pair; SEQ ID NO:31) with thehuman TANGO 228 full length nucleic acid sequence (lower line of eachpair; SEQ ID NO:1). FIGS. 16A-16B show an alignment of the murine TANGO228 coding region (upper line of each pair; SEQ ID NO:32) with the humanTANGO 228 coding region (lower line of each pair; SEQ ID NO:2). FIG. 17shows an alignment of the murine TANGO 228 protein sequence (upper lineof each pair; SEQ ID NO:33) with the human TANGO 228 protein sequence(lower line of each pair; SEQ ID NO:3).

FIGS. 15A-15B and FIGS. 16A-4B show that there is an overall 58.2%identity between the full length murine TANGO 228 nucleic acid moleculeand the full length human TANGO 228 nucleic acid molecule over thelength of the murine molecule and an overall 58.2% identity between theopen reading frame of murine TANGO 228 nucleic acid molecule and theopen reading frame of the human TANGO 228 nucleic acid molecule over thelength of the murine molecule, respectively. The amino acid alignment inFIG. 17 shows a 30.6% overall amino acid sequence identity betweenmurine TANGO 228 and human TANGO 228 over the length of the murinemolecule

Uses of TANGO 228 Nucleic Acids, Polypeptides, and Modulators Thereof

As TANGO 228 was originally found in a fetal spleen library, TANGO 228nucleic acids, proteins, and modulators thereof can be used to modulatethe proliferation, differentiation, and/or function of cells that formthe spleen, e.g., cells of the splenic connective tissue, e.g., splenicsmooth muscle cells and/or endothelial cells of the splenic bloodvessels. TANGO 228 nucleic acids, proteins, and modulators thereof canalso be used to modulate the proliferation, differentiation, and/orfunction of cells that are processed, e.g., regenerated or phagocytizedwithin the spleen, e.g., erythrocytes and/or B and T lymphocytes andmacrophages. Thus TANGO 228 nucleic acids, proteins, and modulatorsthereof can be used to treat spleen, e.g., the fetal spleen, associateddiseases and disorders. Examples of splenic diseases and disordersinclude e.g., splenic lymphoma and/or splenomegaly, and/or phagocytoticdisorders, e.g., those inhibiting macrophage engulfment of bacteria andviruses in the bloodstream.

Due to TANGO 228's homology to rat MCA-32, which is a cell surfaceantigen gene up-regulated in activated mast cells, TANGO 228 nucleicacids, proteins, and modulators thereof can be used to modulate mastcell function and thus to treat immunologic diseases and disorders.Examples of immunologic diseases and disorders include allergicdisorders, e.g., anaphylaxis and allergic asthma, and inflammatorydisorders, e.g., atopic dermatitis.

Because TANGO 228 is homologous to rat MCA-32, which is expressed inmonocyte and macrophage cell lines, TANGO 228 nucleic acids, proteins,and modulators thereof can be used to protect the body from antigenicinvaders, e.g., by modulating the activity of macrophages, and can beused to treat allergies, e.g., anaphylactic shock and/or allergicrhinitis.

In addition, as TANGO 228 includes one or more Ig domains, TANGO 228nucleic acids, proteins, and modulators thereof can be used to modulateimmunologic function, e.g., by the modulation of immunoglobulins and theformation of antibodies. For the same reason, TANGO 228 nucleic acids,proteins, and modulators thereof can be used to modulate Type Iimmunologic disorders, e.g., anaphylaxis and/or rhinitis, by modulating,e.g., stabilizing, the interaction between antigens and mast cellreceptors, e.g., high affinity IgE receptors.

Due to its mapping in the same region as TNFAIP1, a tumor necrosisfactor, TANGO 228 nucleic acids, proteins, and modulators thereof can besued to treat TNF related disorders (e.g., acute myocarditis, myocardialinfarction, congestive heart failure, T cell disorders (e.g.,dermatitis, fibrosis)), differentiative and apoptotic disorders, anddisorders related to angiogenesis (e.g., tumor formation and/ormetastasis, cancer). Modulators of TANGO 228 expression and/or activitycan be used to treat such disorders.

Human TANGO 240

A cDNA encoding human TANGO 240 was identified by analyzing thesequences of clones present in an osteoblast cDNA library. This analysisled to the identification of a clone, jthoc087d01, encoding full-lengthhuman TANGO 240. The human TANGO 240 cDNA of this clone is 2165nucleotides long (FIG. 6; SEQ ID NO:13). The open reading frame of thiscDNA, nucleotides 2 to 1126 of SEQ ID NO:13 (SEQ ID NO:14), encodes a374 amino acid secreted protein (FIG. 6; SEQ ID NO:15).

The signal peptide prediction program SIGNALP (Nielsen, et al. (1997)Protein Engineering 10:1-6) predicted that human TANGO 240 includes an34 amino acid signal peptide (amino acid 1 to about amino acid 34 of SEQID NO:15) (SEQ ID NO:17) preceding the mature TANGO 240 protein(corresponding to about amino acid 35 to amino acid 374 of SEQ ID NO:15;SEQ ID NO:16). The TANGO 240 protein molecular weight is 40.6 kDa priorto the cleavage of the signal peptide, 37.2 kDa after cleavage of thesignal peptide.

An N-glycosylation site having the sequence NSTG is found from aminoacids 141 to 144 of SEQ ID NO:15. A cAMP and cGMP-dependent proteinkinase phosphorylation site having the sequence RRVT is found from aminoacids 117 to 120. A protein kinase C phosphorylation site having thesequence SCR is found from amino acids 234 to 236. A second proteinkinase C phosphorylation site having the sequence SGK is found fromamino acids 323 to 325. A casein kinase II phosphorylation site havingthe sequence SNTE is found from amino acids 132 to 135. A second caseinkinase II phosphorylation site having the sequence TEAE is found fromamino acids 147 to 150. A third casein kinase II phosphorylation sitehaving the sequence SWND is found from amino acids 210 to 213. A fourthcasein kinase II phosphorylation site having the sequence TEAE is foundfrom amino acids 227 to 230. A fifth casein kinase II phosphorylationsite having the sequence TGED is found from amino acids 270 to 273. Asixth casein kinase II phosphorylation site having the sequence SVEE isfound from amino acids 311 to 314. A seventh casein kinase IIphosphorylation site having the sequence SGKD is found from amino acids323 to 326. A tyrosine kinase phosphorylation site having the sequenceRVTIDAFY is found from amino acids 118 to 125. An N-myristoylation sitehaving the sequence GLVCGR is found from amino acids 7 to 12. A secondN-myristoylation site having the sequence GAAGSQ is found from aminoacids 30 to 35. A third N-myristoylation site having the sequence GTGAGAis found from amino acids 38 to 43. A fourth N-myristoylation sitehaving the sequence GSLAGS is found from amino acids 44 to 49. A fifthN-myristoylation site having the sequence GAHGNS is found from aminoacids 59 to 64. A sixth N-myristoylation site having the sequence GGLHNRis found from amino acids 237 to 242. A seventh N-myristoylation sitehaving the sequence GQHYAN is found from amino acids 254 to 259. Aneighth N-myristoylation site having the sequence GSYMCH is found fromamino acids 332 to 337. An amidation site having the sequence AGKR isfound from amino acids 221 to 224. A prokaryotic membrane lipoproteinlipid attachment site having the sequence GAGAGSLAGSC is found fromamino acids 40 to 50.

Clone EpT240, which encodes human TANGO 240, was deposited with theAmerican Type Culture Collection (10801 University Boulevard, Manassas,Va. 20110-2209) on Feb. 18, 1999 and assigned Accession Number 207116.This deposit will be maintained under the terms of the Budapest Treatyon the International Recognition of the Deposit of Microorganisms forthe Purposes of Patent Procedure. This deposit was made merely as aconvenience for those of skill in the art and is not an admission that adeposit is required under 35 U.S.C. §112.

FIG. 7 depicts a hydropathy plot of human TANGO 240. Relativelyhydrophobic regions of the protein are shown above the horizontal line,and relatively hydrophilic regions of the protein are below thehorizontal line. The cysteine residues (cys) and potentialN-glycosylation sites (Ngly) are indicated by short vertical lines justbelow the hydropathy trace. The dashed vertical line separates thesignal sequence (amino acids 1 to 34 of SEQ ID NO:15; SEQ ID NO:17) onthe left from the mature protein (amino acids 35 to 374 of SEQ ID NO:15;SEQ ID NO:16) on the right.

Northern analysis of TANGO 240 expression in human tissues showed thatan approximately 2.2 kB transcript is weakly expressed in skeletalmuscle, brain, colon, thymus, spleen, and small intestine, moderatelyexpressed in heart, liver, lung, and peripheral blood leukocytes, andstrongly expressed in kidney and placenta.

Secretion assays indicate that the polypeptide encoded by human TANGO240 is a secreted protein, as the presence of a 48 kD and a 42 kDprotein was detected by performing the assays. The secretion assays wereperformed essentially as follows: 8×10⁵ 293T cells were plated per wellin a 6-well plate and the cells were incubated in growth medium (DMEM,10% fetal bovine serum, penicillin/streptomycin) at 37° C., 5% CO₂overnight. 293T cells were transfected with 2 g of full-length TANGO 240inserted in the pMET7 vector/well and 10 g LipofectAMINE (GIBCO/BRL Cat.#18324-012)/well according to the protocol for GIBCO/BRL LipofectAMINE.The transfectant was removed 5 hours later and fresh growth medium wasadded to allow the cells to recover overnight. The medium was removedand each well was gently washed twice with DMEM without methionine andcysteine (ICN Cat. #16-424-54). Next, 1 ml DMEM without methionine andcysteine with 50 Ci Trans-³⁵S (ICN Cat. #51006) was added to each welland the cells were incubated at 37° C., 5% CO₂ for the appropriate timeperiod. A 150 l aliquot of conditioned medium was obtained and 150 l of2×SDS sample buffer was added to the aliquot. The sample washeat-activated and loaded on a 4-20% SDS-PAGE gel. The gel was fixed andthe presence of secreted protein was detected by autoradiography.

Murine TANGO 240

A cDNA encoding murine TANGO 240 was identified by analyzing thesequences of clones present in a mouse bone marrow stromal cell cDNAlibrary. This analysis led to the identification of a clone,jtmMa100b11, encoding full-length murine TANGO 240. The murine TANGO 240cDNA of this clone is 2426 nucleotides long (FIGS. 18A-18B; SEQ IDNO:39). The open reading frame of this cDNA, nucleotides 2 to 1117 ofSEQ ID NO:39 (SEQ ID NO:40), encodes a 372 amino acid secreted protein(FIGS. 18A-18B; SEQ ID NO:41).

The signal peptide prediction program SIGNALP (Nielsen, et al. (1997)Protein Engineering 10:1-6) predicted that murine TANGO 240 includes a31 amino acid signal peptide (amino acid 1 to about amino acid 31 of SEQID NO:41; SEQ ID NO:43) preceding the mature murine TANGO 240 protein(corresponding to about amino acid 32 to amino acid 372 of SEQ ID NO:11;SEQ ID NO:42). The murine TANGO 240 protein molecular weight is 40.7 kDaprior to the cleavage of the signal peptide, 37.3 kDa after cleavage ofthe signal peptide.

FIGS. 19A-19D show an alignment of the murine TANGO 240 full lengthnucleic acid sequence (upper line of each pair; SEQ ID NO:39) with thehuman TANGO 240 full length nucleic acid sequence (lower line of eachpair; SEQ ID NO:13). FIGS. 20A-20B show an alignment of the murine TANGO240 coding region (upper line of each pair; SEQ ID NO:40) with the humanTANGO 240 coding region (lower line of each pair; SEQ ID NO:14). FIG. 21shows an alignment of the murine TANGO 240 protein sequence (upper lineof each pair; SEQ ID NO:41) with the human TANGO 240 protein sequence(lower line of each pair; SEQ ID NO:15).

FIGS. 19A-19D and FIGS. 20A-20B show that there is an overall 78.4%identity between the full length murine TANGO 240 nucleic acid moleculeand the full length human TANGO 240 nucleic acid molecule, over thelength of the human molecule, and an overall 84.4% identity between theopen reading frame of murine TANGO 240 nucleic acid molecule and theopen reading frame of the human TANGO 240 nucleic acid molecule,respectively. The amino acid alignment in FIG. 21 shows an 86% overallamino acid sequence identity between murine TANGO 240 and human TANGO240.

Northern analysis of TANGO 240 expression in mouse tissues showed thatan approximately 2.2 kB transcript is weakly expressed in spleen andskeletal muscle, moderately expressed in testis, and strongly expressedin heart, brain, lung, liver, and kidney.

Uses of TANGO 240 Nucleic acids, Polypeptides, and Modulators Thereof

As TANGO 240 was originally found in an osteoblast library, TANGO 240nucleic acids, proteins, and modulators thereof can be used to modulatethe proliferation, differentiation, and/or function of bone andcartilage cells, e.g., chondrocytes and osteoblasts, and to treat boneand/or cartilage associated diseases or disorders. Examples of boneand/or cartilage diseases and disorders include bone and/or cartilageinjury due to for example, trauma (e.g., bone breakage, cartilagetearing), degeneration (e.g., osteoporosis), degeneration of joints,e.g., arthritis, e.g., osteoarthritis, and bone wearing.

As TANGO 240 is homologous to Mycobacterium tuberculosis conservedhypothetical protein Rv0712, TANGO 240 nucleic acids, proteins, andmodulators thereof can be used to treat diseases associated withbacterial infection, e.g., tuberculosis, e.g., pulmonary tuberculosis.In addition, TANGO 240 can be used to modulate, e.g., trigger, theimmune response and can be used to treat immunologic disease anddisorders, e.g., those associated with the respiratory system, e.g.,asthma.

TANGO 240 nucleic acids, proteins, and modulators thereof can also beused to treat disorders of the cells and tissues in which it isexpressed. As TANGO 240 is expressed in heart, brain, spleen, lung,liver, skeletal muscle, kidney, testis, colon, thymus, peripheral bloodleukocytes, small intestine, and placenta, TANGO 240 nucleic acids,proteins, and modulators thereof can be used to treat disorders of thesecells, tissues, or organs, e.g., ischemic heart disease oratherosclerosis, head trauma, brain cancer, splenic lymphoma,splenomegaly, lung cancer, cystic fibrosis, rheumatoid lung disease,liver cirrhosis, hepatitis, muscular dystrophy, stroke, muscularatrophy, glomerulonephritis, end stage renal disease, uremia, testicularcancer, colon cancer and colonic volvulus, DiGeorge syndrome, thymoma,autoimmune disorders, atresia, Crohns disease, and various placentaldisorders.

Human TANGO 243

A cDNA encoding human TANGO 243 was identified by analyzing thesequences of clones present in a fetal spleen cDNA library. Thisanalysis led to the identification of a clone, jthsa049g04, encodingpotentially full-length human TANGO 243. The human TANGO 243 cDNA ofthis clone is 2811 nucleotides long (FIG. 9; SEQ ID NO:19). The openreading frame of this cDNA, nucleotides 183 to 2567 of SEQ ID NO:19 (SEQID NO:20), encodes a 795 amino acid protein (FIG. 9; SEQ ID NO:21). TheTANGO 243 protein molecular weight is 87.1 kDa.

Like TANGO 243, proteins with a WD domain contain a number of conservedG-beta repeats and are intracellular. The functions associated with WDdomains and G-beta repeats, e.g., signal transduction, cell divisioncontrol, transcriptional regulation, vesicular trafficking, areperformed within the cell.

TANGO 243 includes 6 G-beta domains and one G-beta-like domain. G-betadomain 1 is located at about amino acids 8 to 47 of SEQ ID NO:21 (SEQ IDNO:22). G-beta domain 2 is located at about amino acids 55 to 98 of SEQID NO:21 (SEQ ID NO:23). G-beta domain 3 is located at about amino acids102 to 139 of SEQ ID NO:21 (SEQ ID NO:24). G-beta domain 4 is located atabout amino acids 141 to 179 of SEQ ID NO:21 (SEQ ID NO:25). G-betadomain 5 is located at about amino acids 181 to 218 of SEQ ID NO:21 (SEQID NO:26). G-beta domain 6 is located at about amino acids 221 to 259 ofSEQ ID NO:21 (SEQ ID NO:27). The G-beta-like domain is located at aboutamino acids 261 to 298 of SEQ ID NO:21 (SEQ ID NO:28).

An N-glycosylation site having the sequence NRSF is found from aminoacids 52 to 55 of SEQ ID NO:21. A second N-glycosylation site having thesequence NTSD is found from amino acids 421 to 424. A thirdN-glycosylation site having the sequence NGTA is found from amino acids559 to 562. A fourth N-glycosylation site having the sequence NSSS isfound from amino acids 585 to 588. A fifth N-glycosylation site havingthe sequence NYSV is found from amino acids 708 to 711. A cAMP andcGMP-dependent protein kinase phosphorylation site having the sequenceKKLT is found from amino acids 566 to 569. A second cAMP andcGMP-dependent protein kinase phosphorylation site having the sequenceKKYS is found from amino acids 773 to 776. A protein kinase Cphosphorylation site having the sequence SLR is found from amino acids13 to 15. A second protein kinase C phosphorylation site having thesequence TTR is found from amino acids 42 to 44. A third protein kinaseC phosphorylation site having the sequence SGK is found from amino acids120 to 122. A fourth protein kinase C phosphorylation site having thesequence TAK is found from amino acids 134 to 136. A fifth proteinkinase C phosphorylation site having the sequence TVK is found fromamino acids 174 to 176. A sixth protein kinase C phosphorylation sitehaving the sequence SIR is found from amino acids 213 to 215. A seventhprotein kinase C phosphorylation site having the sequence SLR is foundfrom amino acids 254 to 256. An eighth protein kinase C phosphorylationsite having the sequence TIR is found from amino acids 266 to 268. Aninth protein kinase C phosphorylation site having the sequence SGK isfound from amino acids 391 to 393. A tenth protein kinase Cphosphorylation site having the sequence SYK is found from amino acids415 to 417. An eleventh protein kinase C phosphorylation site having thesequence SEK is found from amino acids 588 to 590. A twelfth proteinkinase C phosphorylation site having the sequence SIK is found fromamino acids 620 to 622. A thirteenth protein kinase C phosphorylationsite having the sequence SQR is found from amino acids 678 to 680. Afourteenth protein kinase C phosphorylation site having the sequence SNKis found from amino acids 694 to 696. A fifteenth protein kinase Cphosphorylation site having the sequence TFR is found from amino acids742 to 744. A casein kinase II phosphorylation site having the sequenceSFTE is found from amino acids 54 to 57. A second casein kinase IIphosphorylation site having the sequence SGHE is found from amino acids188 to 191. A third casein kinase II phosphorylation site having thesequence SETE is found from amino acids 201 to 204. A fourth caseinkinase II phosphorylation site having the sequence TTAE is found fromamino acids 248 to 251. A fifth casein kinase II phosphorylation sitehaving the sequence TESE is found from amino acids 298 to 301. A sixthcasein kinase II phosphorylation site having the sequence SAEE is foundfrom amino acids 306 to 309. A seventh casein kinase II phosphorylationsite having the sequence SVSE is found from amino acids 368 to 371. Aneighth casein kinase II phosphorylation site having the sequence TSDD isfound from amino acids 422 to 425. A ninth casein kinase IIphosphorylation site having the sequence SFSD is found from amino acids466 to 469. A tenth casein kinase II phosphorylation site having thesequence TAPE is found from amino acids 561 to 564. An eleventh caseinkinase II phosphorylation site having the sequence TEDD is found fromamino acids 569 to 572. A twelfth casein kinase II phosphorylation sitehaving the sequence SSSE is found from amino acids 586 to 589. Athirteenth casein kinase II phosphorylation site having the sequenceSVNE is found from amino acids 625 to 628. A fourteenth casein kinase IIphosphorylation site having the sequence SQRE is found from amino acids678 to 681. A fifteenth casein kinase II phosphorylation site having thesequence TILE is found from amino acids 731 to 734. A sixteenth caseinkinase II phosphorylation site having the sequence SVSE is found fromamino acids 777 to 780. An N-myristoylation site having the sequenceGLVCCA is found from amino acids 23 to 28. A second N-myristoylationsite having the sequence GAFVSV is found from amino acids 32 to 37. Athird N-myristoylation site having the sequence GLIATG is found fromamino acids 83 to 88. A fourth N-myristoylation site having the sequenceGNDHNI is found from amino acids 89 to 94. A fifth N-myristoylation sitehaving the sequence GTLLSG is found from amino acids 124 to 129. A sixthN-myristoylation site having the sequence GLMLTG is found from aminoacids 164 to 169. A seventh N-myristoylation site having the sequenceGASDGI is found from amino acids 288 to 293. An eighth N-myristoylationsite having the sequence GTREGQ is found from amino acids 347 to 352. Aninth N-myristoylation site having the sequence GSSGAN is found fromamino acids 382 to 387. A tenth N-myristoylation site having thesequence GQMLGL is found from amino acids 457 to 462. An eleventhN-myristoylation site having the sequence GSSGSS is found from aminoacids 480 to 485. A twelfth N-myristoylation site having the sequenceGTTMAG is found from amino acids 508 to 513. A thirteenthN-myristoylation site having the sequence GAQFSS is found from aminoacids 636 to 641. A fourteenth N-myristoylation site having the sequenceGSNKNI is found from amino acids 693 to 698. A fifteenthN-myristoylation site having the sequence GTLISD is found from aminoacids 750 to 755.

Clone EpT243, which encodes human TANGO 243, was deposited with theAmerican Type Culture Collection (10801 University Boulevard, Manassas,Va. 20110-2209) on Feb. 18, 1999 and assigned Accession Number 207116.This deposit will be maintained under the terms of the Budapest Treatyon the International Recognition of the Deposit of Microorganisms forthe Purposes of Patent Procedure. This deposit was made merely as aconvenience for those of skill in the art and is not an admission that adeposit is required under 35 U.S.C. §112.

FIG. 10 depicts a hydropathy plot of human TANGO 243. Relativelyhydrophobic regions of the protein are shown above the horizontal line,and relatively hydrophilic regions of the protein are below thehorizontal line. The cysteine residues (cys) and potentialN-glycosylation sites (Ngly) are indicated by short vertical lines justbelow the hydropathy trace.

TANGO 243 bears some similarity to human PLAP (GenBank Accession NumberAF083395). An alignment of the nucleotide sequence of human PLAP (SEQ IDNO:29; GenBank Accession Number AF083395) and the nucleotide sequence ofhuman TANGO 243 (SEQ ID NO:19) is depicted in FIGS. 11A-11F. In thisalignment, the nucleotide sequences of human PLAP and human TANGO 243are 78.8% identical. This alignment was performed using the ALIGNalignment program with a PAM120 scoring matrix, a gap length penalty of12, and a gap penalty of 4. FIGS. 12A-12E depict an alignment of thenucleotide sequence of the open reading frames of human PLAP(nucleotides 1 to 2217 of SEQ ID NO:29) and human TANGO 243 (SEQ IDNO:20). The nucleotide sequences of the open reading frames of humanPLAP and human TANGO 243 (SEQ ID NO:20) are 92.7% identical. Thisalignment was performed using the ALIGN alignment program with a PAM120scoring matrix, a gap length penalty of 12, and a gap penalty of 4. Analignment of the amino acid sequence of human PLAP (SEQ ID NO:30) andthe amino acid sequence of human TANGO 243 (SEQ ID NO:21) is depicted inFIGS. 13A-13B. The amino acid sequences of human PLAP and human TANGO243 are 92.8% identical. This alignment was performed using the ALIGNalignment program with a PAM120 scoring matrix, a gap length penalty of12, and a gap penalty of 4.

It is possible that the human TANGO 243 clone described above,jthsa049g04, is a less than full-length clone and encodes a portion ofhuman TANGO 243. If so, the start site of translation is locatedupstream of the 5′ end of this clone. FIG. 22 depicts an alternativetranslation of the 2811 nucleotide long human TANGO 243 cDNA clonedescribed above. In this translation, the open reading frame of thecDNA, nucleotides 3 to 2567 of SEQ ID NO:19 (SEQ ID NO:44), encodes a885 amino acid protein (FIG. 21; SEQ ID NO:45). The molecular weight ofthis TANGO 243 protein is 93.7 kDa.

In this alternative translation of the human TANGO 243, G-beta domain 1is located at about amino acids 68 to 107 of SEQ ID NO:45 (SEQ IDNO:22), G-beta domain 2 is located at about amino acids 115 to 158 ofSEQ ID NO:45 (SEQ ID NO:23), G-beta domain 3 is located at about aminoacids 162 to 199 of SEQ ID NO:45 (SEQ ID NO:24), G-beta domain 4 islocated at about amino acids 201 to 239 of SEQ ID NO:45 (SEQ ID NO:25),G-beta domain 5 is located at about amino acids 241 to 278 of SEQ IDNO:45 (SEQ ID NO:26), G-beta domain 6 is located at about amino acids281 to 319 of SEQ ID NO:45 (SEQ ID NO:27), and the G-beta-like domain islocated at about amino acids 321 to 358 of SEQ ID NO:45 (SEQ ID NO:28).

Within this longer form of TANGO 243, there are regions withconsiderable similarity to human PLAP. For example, a region of humanTANGO 243 from amino acids 358 to 609 of SEQ ID NO:45 (SEQ ID NO:47)constitute PLAP-like domain 1. A region of human TANGO 243 from aminoacids 528 to 758 of SEQ ID NO:45 (SEQ ID NO:48) constitute PLAP-likedomain 2. A region of human TANGO 243 from amino acids 118 to 205 of SEQID NO:45 (SEQ ID NO:49) constitute PLAP-like domain 3. A region of humanTANGO 243 from amino acids 239 to 273 of SEQ ID NO:45 (SEQ ID NO:50)constitute PLAP-like domain 4.

In this alternative translation of the TANGO 243 clone the followingsites are present. An N-glycosylation site having the sequence NRSF isfound from amino acids 112 to 115 of SEQ ID NO:45. A secondN-glycosylation site having the sequence NTSD is found from amino acids481 to 484. A third N-glycosylation site having the sequence NGTA isfound from amino acids 619 to 622. A fourth N-glycosylation site havingthe sequence NSSS is found from amino acids 645 to 648. A fifthN-glycosylation site having the sequence NYSV is found from amino acids768 to 771. A cAMP and cGMP-dependent protein kinase phosphorylationsite having the sequence KKLT is found from amino acids 626 to 629. Asecond cAMP and cGMP-dependent protein kinase phosphorylation sitehaving the sequence KKYS is found from amino acids 833 to 836. A proteinkinase C phosphorylation site having the sequence SGR is found fromamino acids 22 to 24. A second protein kinase C phosphorylation sitehaving the sequence SLR is found from amino acids 73 to 75. A thirdprotein kinase C phosphorylation site having the sequence TTR is foundfrom amino acids 102 to 104. A fourth protein kinase C phosphorylationsite having the sequence SGK is found from amino acids 180 to 182. Afifth protein kinase C phosphorylation site having the sequence TAK isfound from amino acids 194 to 196. A sixth protein kinase Cphosphorylation site having the sequence TVK is found from amino acids234 to 236. A seventh protein kinase C phosphorylation site having thesequence SIR is found from amino acids 273 to 275. An eighth proteinkinase C phosphorylation site having the sequence SLR is found fromamino acids 314 to 316. A ninth protein kinase C phosphorylation sitehaving the sequence TIR is found from amino acids 326 to 328. A tenthprotein kinase C phosphorylation site having the sequence SGK is foundfrom amino acids 451 to 453. An eleventh protein kinase Cphosphorylation site having the sequence SYK is found from amino acids475 to 477. A twelfth protein kinase C phosphorylation site having thesequence SEK is found from amino acids 648 to 650. A thirteenth proteinkinase C phosphorylation site having the sequence SIK is found fromamino acids 680 to 682. A fourteenth protein kinase C phosphorylationsite having the sequence SQR is found from amino acids 738 to 740. Afifteenth protein kinase C phosphorylation site having the sequence SNKis found from amino acids 754 to 756. A sixteenth protein kinase Cphosphorylation site having the sequence TFR is found from amino acids802 to 804. A casein kinase II phosphorylation site having the sequenceSFTE is found from amino acids 114 to 117. A second casein kinase IIphosphorylation site having the sequence SGHE is found from amino acids248 to 252. A third casein kinase II phosphorylation site having thesequence SETE is found from amino acids 261 to 264. A fourth caseinkinase II phosphorylation site having the sequence TTAE is found fromamino acids 308 to 331. A fifth casein kinase II phosphorylation sitehaving the sequence TESE is found from amino acids 358 to 361. A sixthcasein kinase II phosphorylation site having the sequence SAEE is foundfrom amino acids 366 to 369. A seventh casein kinase II phosphorylationsite having the sequence SVSE is found from amino acids 428 to 431. Aneighth casein kinase II phosphorylation site having the sequence TSDD isfound from amino acids 482 to 485. A ninth casein kinase IIphosphorylation site having the sequence SFSD is found from amino acids526 to 529. A tenth casein kinase II phosphorylation site having thesequence TAPE is found from amino acids 621 to 624. An eleventh caseinkinase II phosphorylation site having the sequence TEDD is found fromamino acids 629 to 632. A twelfth casein kinase II phosphorylation sitehaving the sequence SSSE is found from amino acids 646 to 649. Athirteenth casein kinase II phosphorylation site having the sequenceSVNE is found from amino acids 685 to 688. A fourteenth casein kinase IIphosphorylation site having the sequence SQRE is found from amino acids738 to 741. A fifteenth casein kinase II phosphorylation site having thesequence TILE is found from amino acids 791 to 794. A sixteenth caseinkinase II phosphorylation site having the sequence SVSE is found fromamino acids 837 to 840. An N-myristoylation site having the sequenceGSSPAA is found from amino acids 29 to 34. A second N-myristoylationsite having the sequence GARQTL is found from amino acids 54 to 59. Athird N-myristoylation site having the sequence GLVCCA is found fromamino acids 83 to 88. A fourth N-myristoylation site having the sequenceGAFVSV is found from amino acids 92 to 97. A fifth N-myristoylation sitehaving the sequence GLIATG is found from amino acids 83 to 88. A sixthN-myristoylation site having the sequence GNDHNI is found from aminoacids 149 to 154. A seventh N-myristoylation site having the sequenceGTLLSG is found from amino acids 184 to 189. An eighth N-myristoylationsite having the sequence GLMLTG is found from amino acids 224 to 229. Aninth N-myristoylation site having the sequence GASDGI is found fromamino acids 348 to 353. An tenth N-myristoylation site having thesequence GTREGQ is found from amino acids 407 to 412. A eleventhN-myristoylation site having the sequence GSSGAN is found from aminoacids 442 to 447. A twelfth N-myristoylation site having the sequenceGQMLGL is found from amino acids 517 to 522. A thirteenthN-myristoylation site having the sequence GSSGSS is found from aminoacids 540 to 545. A fourteenth N-myristoylation site having the sequenceGTTMAG is found from amino acids 568 to 573. A fifteenthN-myristoylation site having the sequence GAQFSS is found from aminoacids 696 to 701. A sixteenth N-myristoylation site having the sequenceGSNKNI is found from amino acids 753 to 758. A seventeenthN-myristoylation site having the sequence GTLISD is found from aminoacids 810 to 815.

FIG. 23 depicts a hydropathy plot of the 255 amino acid human TANGO 243protein arising from the alternative translation. Relatively hydrophobicregions of the protein are shown above the horizontal line, andrelatively hydrophilic regions of the protein are below the horizontalline. The cysteine residues (cys) and potential N-glycosylation sites(Ngly) are indicated by short vertical lines just below the hydropathytrace.

Uses of TANGO 243 Nucleic Acids, Polypeptides, and Modulators Thereof

As TANGO 243 was originally found in a fetal spleen library, TANGO 243nucleic acids, proteins, and modulators thereof can be used to modulatethe proliferation, differentiation, and/or function of cells that formthe spleen, e.g., cells of the splenic connective tissue, e.g., splenicsmooth muscle cells and/or endothelial cells of the splenic bloodvessels. TANGO 243 nucleic acids, proteins, and modulators thereof canalso be used to modulate the proliferation, differentiation, and/orfunction of cells that are processed, e.g., regenerated or phagocytizedwithin the spleen, e.g., erythrocytes and/or B and T lymphocytes andmacrophages. Thus TANGO 243 nucleic acids, proteins, and modulatorsthereof can be used to treat spleen, e.g., the fetal spleen, associateddiseases and disorders. Examples of splenic diseases and disordersinclude e.g., splenic lymphoma and/or splenomegaly, and/or phagocytoticdisorders, e.g., those inhibiting macrophage engulfment of bacteria andviruses in the bloodstream.

Since TANGO 243 is homologous to human PLAP, which is up-regulated inpatients with rheumatoid arthritis, and which activates moleculesinvolved in the arthropathy pathway, TANGO 243 nucleic acids, proteins,and modulators thereof can be used to treat inflammatory arthropathy,and in bone and cartilage diseases and disorders, e.g., bone andcartilage degenerative diseases and disorders, e.g., arthritis, e.g.,rheumatoid arthritis.

Because both TANGO 243 and PLAP hydrolyze phospholipids intolysophospholipids, which are known to modulate chemotaxis, regulateblood vessel permeability, and promote membrane fusion, and can causemonocytes to secrete IL-1 (interleukin 1), in turn modulatingT-cell-mediated immunity, TANGO 243 nucleic acids, proteins, andmodulators thereof can be used to modulate chemotaxis, regulate bloodvessel permeability, and promote membrane fusion, and can causemonocytes to secrete IL-1 (interleukin 1), in turn modulatingT-cell-mediated immunity.

TANGO 243 and PLAP can also be used to modulate IL-1 and TNF (tumornecrosis factor) synthesis and release (e.g., by modulating, e.g.,activating PLA2 (phospholipase A2), which releases arachidonic acid as abyproduct of hydrolysis, which regulates IL-1 and TNF transcription). AsIL-1 and TNF are involved in immunologic processes, TANGO 243 nucleicacids, proteins, and modulators thereof can be used to modulate suchprocesses and to treat immunologic disorders.

By activating PLA2, TANGO 243 nucleic acids, proteins, and modulatorsthereof can modulate levels of arachidonic acid, and thus control levelsof eicosanoids and prostaglandins. By controlling the levels ofeicosanoids and prostaglandins, TANGO 243 nucleic acids, proteins, andmodulators thereof can modulate the immunologic pathway.

The presence of one or more G-beta domain suggests that TANGO 228functions in a manner similar to other G-beta-containing proteins, suchas members of the WD-repeat family. For example, members of WD-repeatfamily typically have biological functions that include, but are notlimited to, signal transduction, cell divisional control, transcriptionregulation, and vesicular trafficking.

Tables 1 and 2 below provide a summary of the sequence information forhuman is TANGO 228, human TANGO 240, and human TANGO 243. Tables 3 and 4below provide a summary of the sequence information for murine TANGO 228and murine TANGO 240.

TABLE 1 Summary of Human TANGO 228, TANGO 240 and TANGO 243 SequenceInformation Accession Gene cDNA ORF FIG. Number TANGO 228 SEQ ID NO: 1SEQ ID NO: 2 FIGS. 1A-1C 207116 TANGO 240 SEQ ID NO: 13 SEQ ID NO: 14FIGS. 6A-6B 207116 TANGO 243 SEQ ID NO: 19 SEQ ID NO: 20 FIGS. 9A-9C207116 TANGO 243 SEQ ID NO: 19 SEQ ID NO: 44 FIGS. 22A-22C 207116Alternative translation

TABLE 2 Summary of Domains of Human TANGO 228, TANGO 240, and TANGO 243Proteins Signal Mature Protein Sequence Protein Extracellular IgTransmembrane Cytoplasmic G-beta TANGO aa 1-19 aa 20-343 aa 1-227 aa 49-aa 228-249 aa 250- 228 of of SEQ ID of 105 and of SEQ ID 343 of SEQ IDNO: 3 SEQ ID 140-198 NO: 3 SEQ ID NO: 3 (SEQ ID NO: 3 of (SEQ ID NO: 3(SEQ ID NO: 4) (SEQ ID SEQ ID NO: 7) (SEQ ID NO: 5) NO: 6) NO: 3 NO: 10)(SEQ ID NO: 8 and 9) TANGO aa 1-34 aa 35-374 240 of of SEQ ID SEQ ID NO:13 NO: 13 (SEQ ID (SEQ ID NO: 16) NO: 17) TANGO aa 8-47, 243 55-98,102-139, 141-179, 181-218, 221-259, and 261- 298 of SEQ ID NO: 19 (SEQID NO: 22-28) TANGO aa 68-107, 243 115-158, Alternative 162-199,Translation 201-239, 241-278, 281-319, and 321- 358 of SEQ ID NO: 45(SEQ ID NO: 22-28)

TABLE 3 Summary of Murine TANGO 228 and Murine TANGO 240 SequenceInformation Accession Gene cDNA ORF FIG. Number TANGO 228 SEQ ID NO: 31SEQ ID NO: 32 FIG. 14 TANGO 240 SEQ ID NO: 43 SEQ ID NO: 44 FIG. 18

TABLE 4 Summary of Domains of Murine TANGO 228 and TANGO 240 ProteinsSignal Mature Protein Sequence Protein Extracellular TransmembraneCytoplasmic TANGO aa 1-34 aa 35-239 aa 35-141 aa 142-164 aa 165- 228 ofof SEQ ID of of SEQ ID 239 of SEQ ID NO: 33 SEQ ID NO: 33 SEQ ID NO: 33(SEQ ID NO: 33 (SEQ ID NO: 33 (SEQ ID NO: 34) (SEQ ID NO: 37) (SEQ IDNO: 35) NO: 36) NO: 38) TANGO aa 1-31 aa 32-372 240 of of SEQ ID SEQ IDNO: 41 NO: 41 (SEQ ID (SEQ ID NO: 42) NO: 43)

Various aspects of the invention are described in further detail in thefollowing subsections:

I. Isolated Nucleic Acid Molecules

One aspect of the invention pertains to isolated nucleic acid moleculesthat encode a polypeptide of the invention or a biologically activeportion thereof, as well as nucleic acid molecules sufficient for use ashybridization probes to identify nucleic acid molecules encoding apolypeptide of the invention and fragments of such nucleic acidmolecules suitable for use as PCR primers for the amplification ormutation of nucleic acid molecules. As used herein, the term “nucleicacid molecule” is intended to include DNA molecules (e.g., cDNA orgenomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA orRNA generated using nucleotide analogs. The nucleic acid molecule can besingle-stranded or double-stranded, but preferably is double-strandedDNA.

An “isolated” nucleic acid molecule is one which is separated from othernucleic acid molecules which are present in the natural source of thenucleic acid molecule. Preferably, an “isolated” nucleic acid moleculeis free of sequences (preferably protein encoding sequences) whichnaturally flank the nucleic acid (i.e., sequences located at the 5′ and3′ ends of the nucleic acid) in the genomic DNA of the organism fromwhich the nucleic acid is derived. For example, in various embodiments,the isolated nucleic acid molecule can contain less than about 5 kB, 4kB, 3 kB, 2 kB, 1 kB, 0.5 kB or 0.1 kB of nucleotide sequences whichnaturally flank the nucleic acid molecule in genomic DNA of the cellfrom which the nucleic acid is derived. Moreover, an “isolated” nucleicacid molecule, such as a cDNA molecule, can be substantially free ofother cellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized. As used herein, the term“isolated” when referring to a nucleic acid molecule does not include anisolated chromosome.

A nucleic acid molecule of the present invention, e.g., a nucleic acidmolecule having the nucleotide sequence of SEQ ID NO:1, 2, 3, 13, 14,19, 20, 31, 32, 39, 40, or 44, or a complement thereof, can be isolatedusing standard molecular biology techniques and the sequence informationprovided herein. Using all or a portion of the nucleic acid sequences ofSEQ ID NO:1, 2, 3, 13, 14, 19, 20, 31, 32, 39, 40, or 44 as ahybridization probe, nucleic acid molecules of the invention can beisolated using standard hybridization and cloning techniques (e.g., asdescribed in Sambrook et al., eds., Molecular Cloning: A LaboratoryManual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989).

A nucleic acid molecule of the invention can be amplified using cDNA,mRNA or genomic DNA as a template and appropriate oligonucleotideprimers according to standard PCR amplification techniques. The nucleicacid so amplified can be cloned into an appropriate vector andcharacterized by DNA sequence analysis. Furthermore, oligonucleotidescorresponding to all or a portion of a nucleic acid molecule of theinvention can be prepared by standard synthetic techniques, e.g., usingan automated DNA synthesizer.

In another preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleic acid molecule which is a complement ofthe nucleotide sequence of SEQ ID NO:1, 2, 3, 13, 14, 19, 20, 31, 32,39, 40, or 44, or a portion thereof. A nucleic acid molecule which iscomplementary to a given nucleotide sequence is one which issufficiently complementary to the given nucleotide sequence that it canhybridize to the given nucleotide sequence thereby forming a stableduplex.

Moreover, a nucleic acid molecule of the invention can comprise only aportion of a nucleic acid sequence encoding a full length polypeptide ofthe invention for example, a fragment which can be used as a probe orprimer or a fragment encoding a biologically active portion of apolypeptide of the invention. The nucleotide sequence determined fromthe cloning one gene allows for the generation of probes and primersdesigned for use in identifying and/or cloning homologues in other celltypes, e.g., from other tissues, as well as homologues from othermammals. The probe/primer typically comprises substantially purifiedoligonucleotide. The oligonucleotide typically comprises a region ofnucleotide sequence that hybridizes under stringent conditions to atleast about 12, preferably about 25, more preferably about 50, 75, 100,125, 150, 175, 200, 250, 300, 350 or 400 consecutive nucleotides of thesense or anti-sense sequence of SEQ ID NO:1, 2, 3, 13, 14, 19, 20, 31,32, 39, 40, or 44 or of a naturally occurring mutant of SEQ ID NO:1, 2,3, 13, 14, 19, 20, 31, 32, 39, 40, or 44.

Probes based on the sequence of a nucleic acid molecule of the inventioncan be used to detect transcripts or genomic sequences encoding the sameprotein molecule encoded by a selected nucleic acid molecule. The probecomprises a label group attached thereto, e.g., a radioisotope, afluorescent compound, an enzyme, or an enzyme co-factor. Such probes canbe used as part of a diagnostic test kit for identifying cells ortissues which mis-express the protein, such as by measuring levels of anucleic acid molecule encoding the protein in a sample of cells from asubject, e.g., detecting mRNA levels or determining whether a geneencoding the protein has been mutated or deleted.

A nucleic acid fragment encoding a “biologically active portion” of apolypeptide of the invention can be prepared by isolating a portion ofany of SEQ ID NO:2, 14, or 20, expressing the encoded portion of thepolypeptide protein (e.g., by recombinant expression in vitro) andassessing the activity of the encoded portion of the polypeptide.

The invention further encompasses nucleic acid molecules that differfrom the nucleotide sequence of SEQ ID NO:1, 2, 3, 13, 14, 19, 20, 31,32, 39, 40, or 44, due to degeneracy of the genetic code and thus encodethe same protein as that encoded by the nucleotide sequence of SEQ IDNO:2, 14, or 20.

In addition to the nucleotide sequences of SEQ ID NO:2, 14, and 20, itwill be appreciated by those skilled in the art that DNA sequencepolymorphisms that lead to changes in the amino acid sequence may existwithin a population (e.g., the human population). Such geneticpolymorphisms may exist among individuals within a population due tonatural allelic variation. An allele is one of a group of genes whichoccur alternatively at a given genetic locus. As used herein, the phrase“allelic variant” refers to a nucleotide sequence which occurs at agiven locus or to a polypeptide encoded by the nucleotide sequence. Asused herein, the terms “gene” and “recombinant gene” refer to nucleicacid molecules comprising an open reading frame encoding a polypeptideof the invention. Such natural allelic variations can typically resultin 1-5% variance in the nucleotide sequence of a given gene. Alternativealleles can be identified by sequencing the gene of interest in a numberof different individuals. This can be readily carried out by usinghybridization probes to identify the same genetic locus in a variety ofindividuals. Any and all such nucleotide variations and resulting aminoacid polymorphisms or variations that are the result of natural allelicvariation and that do not alter the functional activity are intended tobe within the scope of the invention.

Moreover, nucleic acid molecules encoding proteins of the invention fromother species (homologues), which have a nucleotide sequence whichdiffers from that of the human protein described herein are intended tobe within the scope of the invention. Nucleic acid moleculescorresponding to natural allelic variants and homologues of a cDNA ofthe invention can be isolated based on their identity to the humannucleic acid molecule disclosed herein using the human cDNAs, or aportion thereof, as a hybridization probe according to standardhybridization techniques under stringent hybridization conditions. Forexample, a cDNA encoding a soluble form of a membrane-bound protein ofthe invention isolated based on its hybridization to a nucleic acidmolecule encoding all or part of the membrane-bound form. Likewise, acDNA encoding a membrane-bound form can be isolated based on itshybridization to a nucleic acid molecule encoding all or part of thesoluble form.

Accordingly, in another embodiment, an isolated nucleic acid molecule ofthe invention is at least 300 (325, 350, 375, 400, 425, 450, 500, 550,600, 650, 700, 800, 900, or 1000) nucleotides in length and hybridizesunder stringent conditions to the nucleic acid molecule comprising thenucleotide sequence, preferably the coding sequence, of SEQ ID NO:1 or13, or a complement thereof.

Accordingly, in another embodiment, an isolated nucleic acid molecule ofthe invention is at least 600 (650, 700, 800, 900, 1000, 1200, 1400,1600, 1800, 2000, or 2200) nucleotides in length and hybridizes understringent conditions to the nucleic acid molecule comprising thenucleotide sequence, preferably the coding sequence, of SEQ ID NO:19, ora complement thereof.

As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences at least 60% (65%, 70%, preferably 75%)identical to each other typically remain hybridized to each other. Suchstringent conditions are known to those skilled in the art and can befound in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.(1989), 6.3.1-6.3.6. A preferred, non-limiting example of stringenthybridization conditions are hybridization in 6× sodium chloride/sodiumcitrate (SSC) at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 50-65° C. Preferably, an isolated nucleic acidmolecule of the invention that hybridizes under stringent conditions tothe sequence of SEQ ID NO:1, 2, 3, 13, 14, 19, 20, 31, 32, 39, 40, or44, or a complement thereof, corresponds to a naturally-occurringnucleic acid molecule. As used herein, a “naturally-occurring” nucleicacid molecule refers to an RNA or DNA molecule having a nucleotidesequence that occurs in nature (e.g., encodes a natural protein).

In addition to naturally-occurring allelic variants of a nucleic acidmolecule of the invention sequence that may exist in the population, theskilled artisan will further appreciate that changes can be introducedby mutation thereby leading to changes in the amino acid sequence of theencoded protein, without altering the biological activity of theprotein. For example, one can make nucleotide substitutions leading toamino acid substitutions at “non-essential” amino acid residues. A“non-essential” amino acid residue is a residue that can be altered fromthe wild-type sequence without altering the biological activity, whereasan “essential” amino acid residue is required for biological activity.For example, amino acid residues that are not conserved or onlysemi-conserved among homologues of various species may be non-essentialfor activity and thus would be likely targets for alteration.Alternatively, amino acid residues that are conserved among thehomologues of various species (e.g., murine and human) may be essentialfor activity and thus would not be likely targets for alteration.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding a polypeptide of the invention that contain changesin amino acid residues that are not essential for activity. Suchpolypeptides differ in amino acid sequence from SEQ ID NO:3, yet retainbiological activity. In one embodiment, the isolated nucleic acidmolecule includes a nucleotide sequence encoding a protein that includesan amino acid sequence that is at least about 30% identical, 35%, 40%,45%, 50%, 55%, 60%, 65%, 75%, 85%, 95%, or 98% identical to the aminoacid sequence of SEQ ID NO:3.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding a polypeptide of the invention that contain changesin amino acid residues that are not essential for activity. Suchpolypeptides differ in amino acid sequence from SEQ ID NO:15, yet retainbiological activity. In one embodiment, the isolated nucleic acidmolecule includes a nucleotide sequence encoding a protein that includesan amino acid sequence that is at least about 35% identical, 40%, 45%,50%, 55%, 65%, 75%, 85%, 95%, or 98% identical to the amino acidsequence of SEQ ID NO:15.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding a polypeptide of the invention that contain changesin amino acid residues that are not essential for activity. Suchpolypeptides differ in amino acid sequence from SEQ ID NO:21, yet retainbiological activity. In one embodiment, the isolated nucleic acidmolecule includes a nucleotide sequence encoding a protein that includesan amino acid sequence that is at least about 93% identical, 94%, 95%,96%, 97% or 98% identical to the amino acid sequence of SEQ ID NO:21.

An isolated nucleic acid molecule encoding a variant protein can becreated by introducing one or more nucleotide substitutions, additionsor deletions into the nucleotide sequence of SEQ ID NO:1, 2, 3, 13, 14,19, 20, 31, 32, 39, 40, or 44 such that one or more amino acidsubstitutions, additions or deletions are introduced into the encodedprotein. Mutations can be introduced by standard techniques, such assite-directed mutagenesis and PCR-mediated mutagenesis. Preferably,conservative amino acid substitutions are made at one or more predictednon-essential amino acid residues. A “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined in the art. Thesefamilies include amino acids with basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Alternatively, mutations can beintroduced randomly along all or part of the coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forbiological activity to identify mutants that retain activity. Followingmutagenesis, the encoded protein can be expressed recombinantly and theactivity of the protein can be determined.

In a preferred embodiment, a mutant polypeptide that is a variant of apolypeptide of the invention can be assayed for: (1) the ability to formprotein:protein interactions with proteins in a signaling pathway of thepolypeptide of the invention; (2) the ability to bind a ligand of thepolypeptide of the invention; or (3) the ability to bind to anintracellular target protein of the polypeptide of the invention. In yetanother preferred embodiment, the mutant polypeptide can be assayed forthe ability to modulate cellular proliferation, cellular migration orchemotaxis, or cellular differentiation.

The present invention encompasses antisense nucleic acid molecules,i.e., molecules which are complementary to a sense nucleic acid encodinga polypeptide of the invention, e.g., complementary to the coding strandof a double-stranded cDNA molecule or complementary to an mRNA sequence.Accordingly, an antisense nucleic acid can hydrogen bond to a sensenucleic acid. The antisense nucleic acid can be complementary to anentire coding strand, or to only a portion thereof, e.g., all or part ofthe protein coding region (or open reading frame). An antisense nucleicacid molecule can be antisense to all or part of a non-coding region ofthe coding strand of a nucleotide sequence encoding a polypeptide of theinvention. The non-coding regions (“5′ and 3′ untranslated regions”) arethe 5′ and 3′ sequences which flank the coding region and are nottranslated into amino acids.

An antisense oligonucleotide can be, for example, about 5, 10, 15, 20,25, 30, 35, 40, 45 or 50 nucleotides or more in length. An antisensenucleic acid of the invention can be constructed using chemicalsynthesis and enzymatic ligation reactions using procedures known in theart. For example, an antisense nucleic acid (e.g., an antisenseoligonucleotide) can be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed between the antisense and sense nucleicacids, e.g., phosphorothioate derivatives and acridine substitutednucleotides can be used. Examples of modified nucleotides which can beused to generate the antisense nucleic acid include 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding a selectedpolypeptide of the invention to thereby inhibit expression, e.g., byinhibiting transcription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule which bindsto DNA duplexes, through specific interactions in the major groove ofthe double helix. An example of a route of administration of antisensenucleic acid molecules of the invention includes direct injection at atissue site. Alternatively, antisense nucleic acid molecules can bemodified to target selected cells and then administered systemically.For example, for systemic administration, antisense molecules can bemodified such that they specifically bind to receptors or antigensexpressed on a selected cell surface, e.g., by linking the antisensenucleic acid molecules to peptides or antibodies which bind to cellsurface receptors or antigens. The antisense nucleic acid molecules canalso be delivered to cells using the vectors described herein. Toachieve sufficient intracellular concentrations of the antisensemolecules, vector constructs in which the antisense nucleic acidmolecule is placed under the control of a strong pol II or pol IIIpromoter are preferred.

An antisense nucleic acid molecule of the invention can be an □-anomericnucleic acid molecule. An □-anomeric nucleic acid molecule formsspecific double-stranded hybrids with complementary RNA in which,contrary to the usual, β-units, the strands run parallel to each other(Gaultier et al. (1987) Nucleic Acids Res. 15:6625-6641). The antisensenucleic acid molecule can also comprise a 2′-o-methylribonucleotide(Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimericRNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).

The invention also encompasses ribozymes. Ribozymes are catalytic RNAmolecules with ribonuclease activity which are capable of cleaving asingle-stranded nucleic acid, such as an mRNA, to which they have acomplementary region. Thus, ribozymes (e.g., hammerhead ribozymes(described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can beused to catalytically cleave mRNA transcripts to thereby inhibittranslation of the protein encoded by the mRNA. A ribozyme havingspecificity for a nucleic acid molecule encoding a polypeptide of theinvention can be designed based upon the nucleotide sequence of a cDNAdisclosed herein. For example, a derivative of a Tetrahymena L-19 IVSRNA can be constructed in which the nucleotide sequence of the activesite is complementary to the nucleotide sequence to be cleaved in a Cechet al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742.Alternatively, an mRNA encoding a polypeptide of the invention can beused to select a catalytic RNA having a specific ribonuclease activityfrom a pool of RNA molecules. See, e.g., Bartel and Szostak (1993)Science 261:1411-1418.

The invention also encompasses nucleic acid molecules which form triplehelical structures. For example, expression of a polypeptide of theinvention can be inhibited by targeting nucleotide sequencescomplementary to the regulatory region of the gene encoding thepolypeptide (e.g., the promoter and/or enhancer) to form triple helicalstructures that prevent transcription of the gene in target cells. Seegenerally Helene (1991) Anticancer Drug Des. 6(6):569-84; Helene (1992)Ann. N.Y. Acad. Sci. 660:27-36; and Maher (1992) Bioassays14(12):807-15.

In various embodiments, the nucleic acid molecules of the invention canbe modified at the base moiety, sugar moiety or phosphate backbone toimprove, e.g., the stability, hybridization, or solubility of themolecule. For example, the deoxyribose phosphate backbone of the nucleicacids can be modified to generate peptide nucleic acids (see Hyrup etal. (1996) Bioorganic & Medicinal Chemistry 4(1): 5-23). As used herein,the terms “peptide nucleic acids” or “PNAs” refer to nucleic acidmimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone isreplaced by a pseudopeptide backbone and only the four naturalnucleobases are retained. The neutral backbone of PNAs has been shown toallow for specific hybridization to DNA and RNA under conditions of lowionic strength. The synthesis of PNA oligomers can be performed usingstandard solid phase peptide synthesis protocols as described in Hyrupet al. (1996), supra; Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci.USA 93: 14670-675.

PNAs can be used in therapeutic and diagnostic applications. Forexample, PNAs can be used as antisense or antigene agents forsequence-specific modulation of gene expression by, e.g., inducingtranscription or translation arrest or inhibiting replication. PNAs canalso be used, e.g., in the analysis of single base pair mutations in agene by, e.g., PNA directed PCR clamping; as artificial restrictionenzymes when used in combination with other enzymes, e.g., S1 nucleases(Hyrup (1996), supra; or as probes or primers for DNA sequence andhybridization (Hyrup (1996), supra; Perry-O'Keefe et al. (1996) Proc.Natl. Acad. Sci. USA 93: 14670-675).

In another embodiment, PNAs can be modified, e.g., to enhance theirstability or cellular uptake, by attaching lipophilic or other helpergroups to PNA, by the formation of PNA-DNA chimeras, or by the use ofliposomes or other techniques of drug delivery known in the art. Forexample, PNA-DNA chimeras can be generated which may combine theadvantageous properties of PNA and DNA. Such chimeras allow DNArecognition enzymes, e.g., RNAse H and DNA polymerases, to interact withthe DNA portion while the PNA portion would provide high bindingaffinity and specificity. PNA-DNA chimeras can be linked using linkersof appropriate lengths selected in terms of base stacking, number ofbonds between the nucleobases, and orientation (Hyrup (1996), supra).The synthesis of PNA-DNA chimeras can be performed as described in Hyrup(1996), supra, and Finn et al. (1996) Nucleic Acids Res. 24(17):3357-63.For example, a DNA chain can be synthesized on a solid support usingstandard phosphoramidite coupling chemistry and modified nucleosideanalogs. Compounds such as 5′-(4-methoxytrityl)amino-5′-deoxy-thymidinephosphoramidite can be used as a link between the PNA and the 5′ end ofDNA (Mag et al. (1989) Nucleic Acids Res. 17:5973-88). PNA monomers arethen coupled in a stepwise manner to produce a chimeric molecule with a5′ PNA segment and a 3′ DNA segment (Finn et al. (1996) Nucleic AcidsRes. 24(17):3357-63). Alternatively, chimeric molecules can besynthesized with a 5′ DNA segment and a 3′ PNA segment (Peterser et al.(1975) Bioorganic Med. Chem. Lett. 5:1119-11124).

In other embodiments, the oligonucleotide may include other appendedgroups such as peptides (e.g., for targeting host cell receptors invivo), or agents facilitating transport across the cell membrane (see,e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556;Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCTPublication No. WO 88/09810) or the blood-brain barrier (see, e.g., PCTPublication No. WO 89/10134). In addition, oligonucleotides can bemodified with hybridization-triggered cleavage agents (see, e.g., Krolet al. (1988) Bio/Techniques 6:958-976) or intercalating agents (see,e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, theoligonucleotide may be conjugated to another molecule, e.g., a peptide,hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

II. Isolated Proteins and Antibodies

One aspect of the invention pertains to isolated proteins, andbiologically active portions thereof, as well as polypeptide fragmentssuitable for use as immunogens to raise antibodies directed against apolypeptide of the invention. In one embodiment, the native polypeptidecan be isolated from cells or tissue sources by an appropriatepurification scheme using standard protein purification techniques. Inanother embodiment, polypeptides of the invention are produced byrecombinant DNA techniques. Alternative to recombinant expression, apolypeptide of the invention can be synthesized chemically usingstandard peptide synthesis techniques.

An “isolated” or “purified” protein or biologically active portionthereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which theprotein is derived, or substantially free of chemical precursors orother chemicals when chemically synthesized. The language “substantiallyfree of cellular material” includes preparations of protein in which theprotein is separated from cellular components of the cells from which itis isolated or recombinantly produced. Thus, protein that issubstantially free of cellular material includes preparations of proteinhaving less than about 30%, 20%, 10%, or 5% (by dry weight) ofheterologous protein (also referred to herein as a “contaminatingprotein”). When the protein or biologically active portion thereof isrecombinantly produced, it is also preferably substantially free ofculture medium, i.e., culture medium represents less than about 20%,10%, or 5% of the volume of the protein preparation. When the protein isproduced by chemical synthesis, it is preferably substantially free ofchemical precursors or other chemicals, i.e., it is separated fromchemical precursors or other chemicals which are involved in thesynthesis of the protein. Accordingly such preparations of the proteinhave less than about 30%, 20%, 10%, 5% (by dry weight) of chemicalprecursors or compounds other than the polypeptide of interest.

Biologically active portions of a polypeptide of the invention includepolypeptides comprising amino acid sequences sufficiently identical toor derived from the amino acid sequence of the protein (e.g., the aminoacid sequence shown in any of SEQ ID NO:3, 4, 15, 16, 21, 22, 33, 34,41, 42, or 45), which include fewer amino acids than the full lengthprotein, and exhibit at least one activity of the correspondingfull-length protein. Typically, biologically active portions comprise adomain or motif with at least one activity of the corresponding protein.A biologically active portion of a protein of the invention can be apolypeptide which is, for example, 10, 25, 50, 100 or more amino acidsin length. Moreover, other biologically active portions, in which otherregions of the protein are deleted, can be prepared by recombinanttechniques and evaluated for one or more of the functional activities ofthe native form of a polypeptide of the invention.

Preferred polypeptides have the amino acid sequence of SEQ ID NO:3, 4,15, 16, 21, 22, 33, 34, 41, 42, or 45. Other useful proteins aresubstantially identical (e.g., at least about 45%, preferably 55%, 65%,75%, 85%, 95%, or 99%) to any of SEQ ID NO: 3, 4, 15, 16, 21, 22, 33,34, 41, 42, or 45, or substantially identical (e.g., at least about 93%,preferably 94%, 95%, 96%, or 99%) to any of SEQ ID NO:21 or 22, andretain the functional activity of the protein of the correspondingnaturally-occurring protein yet differ in amino acid sequence due tonatural allelic variation or mutagenesis.

To determine the percent identity of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in the sequence of a first amino acid ornucleic acid sequence for optimal alignment with a second amino ornucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity=# ofidentical positions/total # of positions (e.g., overlappingpositions)×100). In one embodiment, the two sequences are the samelength.

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm. A preferred, non-limitingexample of a mathematical algorithm utilized for the comparison of twosequences is the algorithm of Karlin and Altschul (1990) Proc. Natl.Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm isincorporated into the NBLAST and XBLAST programs of Altschul, et al.(1990) J. Mol. Biol. 215:403-410. BLAST nucleotide searches can beperformed with the NBLAST program, score=100, wordlength=12 to obtainnucleotide sequences homologous to a nucleic acid molecules of theinvention. BLAST protein searches can be performed with the XBLASTprogram, score=50, wordlength=3 to obtain amino acid sequenceshomologous to a protein molecules of the invention. To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402.Alternatively, PSI-Blast can be used to perform an iterated search whichdetects distant relationships between molecules (Id.). When utilizingBLAST, Gapped BLAST, and PSI-Blast programs, the default parameters ofthe respective programs (e.g., XBLAST and NBLAST) can be used. Seehttp://www.ncbi.nlm.nih.gov. Another preferred, non-limiting example ofa mathematical algorithm utilized for the comparison of sequences is thealgorithm of Myers and Miller, (1988) CABIOS 4:11-17. Such an algorithmis incorporated into the ALIGN program (version 2.0) which is part ofthe GCG sequence alignment software package. When utilizing the ALIGNprogram for comparing amino acid sequences, a PAM120 weight residuetable, a gap length penalty of 12, and a gap penalty of 4 can be used.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, only exact matches are counted.

The invention also provides chimeric or fusion proteins. As used herein,a “chimeric protein” or “fusion protein” comprises all or part(preferably biologically active) of a polypeptide of the inventionoperably linked to a heterologous polypeptide (i.e., a polypeptide otherthan the same polypeptide of the invention). Within the fusion protein,the term “operably linked” is intended to indicate that the polypeptideof the invention and the heterologous polypeptide are fused in-frame toeach other. The heterologous polypeptide can be fused to the N-terminusor C-terminus of the polypeptide of the invention.

One useful fusion protein is a GST fusion protein in which thepolypeptide of the invention is fused to the C-terminus of GSTsequences. Such fusion proteins can facilitate the purification of arecombinant polypeptide of the invention.

In another embodiment, the fusion protein contains a heterologous signalsequence at its N-terminus. For example, the native signal sequence of apolypeptide of the invention can be removed and replaced with a signalsequence from another protein. For example, the gp67 secretory sequenceof the baculovirus envelope protein can be used as a heterologous signalsequence (Current Protocols in Molecular Biology, Ausubel et al., eds.,John Wiley & Sons, 1992). Other examples of eukaryotic heterologoussignal sequences include the secretory sequences of melittin and humanplacental alkaline phosphatase (Stratagene; La Jolla, Calif.). In yetanother example, useful prokaryotic heterologous signal sequencesinclude the phoA secretory signal (Sambrook et al., supra) and theprotein A secretory signal (Pharmacia Biotech; Piscataway, N.J.).

In yet another embodiment, the fusion protein is an immunoglobulinfusion protein in which all or part of a polypeptide of the invention isfused to sequences derived from a member of the immunoglobulin proteinfamily. The immunoglobulin fusion proteins of the invention can beincorporated into pharmaceutical compositions and administered to asubject to inhibit an interaction between a ligand (soluble ormembrane-bound) and a protein on the surface of a cell (receptor), tothereby suppress signal transduction in vivo. The immunoglobulin fusionprotein can be used to affect the bioavailability of a cognate ligand ofa polypeptide of the invention. Inhibition of ligand/receptorinteraction may be useful therapeutically, both for treatingproliferative and differentiative disorders and for modulating (e.g.,promoting or inhibiting) cell survival. Moreover, the immunoglobulinfusion proteins of the invention can be used as immunogens to produceantibodies directed against a polypeptide of the invention in a subject,to purify ligands and in screening assays to identify molecules whichinhibit the interaction of receptors with ligands.

Chimeric and fusion proteins of the invention can be produced bystandard recombinant DNA techniques. In another embodiment, the fusiongene can be synthesized by conventional techniques including automatedDNA synthesizers. Alternatively, PCR amplification of gene fragments canbe carried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and reamplified to generate a chimeric gene sequence (see,e.g., Ausubel et al., supra). Moreover, many expression vectors arecommercially available that already encode a fusion moiety (e.g., a GSTpolypeptide). A nucleic acid encoding a polypeptide of the invention canbe cloned into such an expression vector such that the fusion moiety islinked in-frame to the polypeptide of the invention.

A signal sequence of a polypeptide of the invention (SEQ ID NO:5 and 19)can be used to facilitate secretion and isolation of the secretedprotein or other proteins of interest. Signal sequences are typicallycharacterized by a core of hydrophobic amino acids which are generallycleaved from the mature protein during secretion in one or more cleavageevents. Such signal peptides contain processing sites that allowcleavage of the signal sequence from the mature proteins as they passthrough the secretory pathway. Thus, the invention pertains to thedescribed polypeptides having a signal sequence, as well as to thesignal sequence itself and to the polypeptide in the absence of thesignal sequence (i.e., the cleavage products). In one embodiment, anucleic acid sequence encoding a signal sequence of the invention can beoperably linked in an expression vector to a protein of interest, suchas a protein which is ordinarily not secreted or is otherwise difficultto isolate. The signal sequence directs secretion of the protein, suchas from a eukaryotic host into which the expression vector istransformed, and the signal sequence is subsequently or concurrentlycleaved. The protein can then be readily purified from the extracellularmedium by art recognized methods. Alternatively, the signal sequence canbe linked to the protein of interest using a sequence which facilitatespurification, such as with a GST domain.

In another embodiment, the signal sequences of the present invention canbe used to identify regulatory sequences, e.g., promoters, enhancers,repressors. Since signal sequences are the most amino-terminal sequencesof a peptide, it is expected that the nucleic acids which flank thesignal sequence on its amino-terminal side will be regulatory sequenceswhich affect transcription. Thus, a nucleotide sequence which encodesall or a portion of a signal sequence can be used as a probe to identifyand isolate signal sequences and their flanking regions, and theseflanking regions can be studied to identify regulatory elements therein.

The present invention also pertains to variants of the polypeptides ofthe invention. Such variants have an altered amino acid sequence whichcan function as either agonists (mimetics) or as antagonists. Variantscan be generated by mutagenesis, e.g., discrete point mutation ortruncation. An agonist can retain substantially the same, or a subset,of the biological activities of the naturally occurring form of theprotein. An antagonist of a protein can inhibit one or more of theactivities of the naturally occurring form of the protein by, forexample, competitively binding to a downstream or upstream member of acellular signaling cascade which includes the protein of interest. Thus,specific biological effects can be elicited by treatment with a variantof limited function. Treatment of a subject with a variant having asubset of the biological activities of the naturally occurring form ofthe protein can have fewer side effects in a subject relative totreatment with the naturally occurring form of the protein.

Variants of a protein of the invention which function as either agonists(mimetics) or as antagonists can be identified by screeningcombinatorial libraries of mutants, e.g., truncation mutants, of theprotein of the invention for agonist or antagonist activity. In oneembodiment, a variegated library of variants is generated bycombinatorial mutagenesis at the nucleic acid level and is encoded by avariegated gene library. A variegated library of variants can beproduced by, for example, enzymatically ligating a mixture of syntheticoligonucleotides into gene sequences such that a degenerate set ofpotential protein sequences is expressible as individual polypeptides,or alternatively, as a set of larger fusion proteins (e.g., for phagedisplay). There are a variety of methods which can be used to producelibraries of potential variants of the polypeptides of the inventionfrom a degenerate oligonucleotide sequence. Methods for synthesizingdegenerate oligonucleotides are known in the art (see, e.g., Narang(1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem.53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983)Nucleic Acid Res. 11:477).

In addition, libraries of fragments of the coding sequence of apolypeptide of the invention can be used to generate a variegatedpopulation of polypeptides for screening and subsequent selection ofvariants. For example, a library of coding sequence fragments can begenerated by treating a double stranded PCR fragment of the codingsequence of interest with a nuclease under conditions wherein nickingoccurs only about once per molecule, denaturing the double stranded DNA,renaturing the DNA to form double stranded DNA which can includesense/antisense pairs from different nicked products, removing singlestranded portions from reformed duplexes by treatment with S1 nuclease,and ligating the resulting fragment library into an expression vector.By this method, an expression library can be derived which encodesN-terminal and internal fragments of various sizes of the protein ofinterest.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.The most widely used techniques, which are amenable to high through-putanalysis, for screening large gene libraries typically include cloningthe gene library into replicable expression vectors, transformingappropriate cells with the resulting library of vectors, and expressingthe combinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recursive ensemble mutagenesis (REM), a techniquewhich enhances the frequency of functional mutants in the libraries, canbe used in combination with the screening assays to identify variants ofa protein of the invention (Arkin and Yourvan (1992) Proc. Natl. Acad.Sci. USA 89:7811-7815; Delgrave et al. (1993) Protein Engineering6(3):327-331).

An isolated polypeptide of the invention, or a fragment thereof, can beused as an immunogen to generate antibodies using standard techniquesfor polyclonal and monoclonal antibody preparation. The full-lengthpolypeptide or protein can be used or, alternatively, the inventionprovides antigenic peptide fragments for use as immunogens. Theantigenic peptide of a protein of the invention comprises at least 8(preferably 10, 15, 20, or 30) amino acid residues of the amino acidsequence of SEQ ID NO:3, 15, 21, or 45, and encompasses an epitope ofthe protein such that an antibody raised against the peptide forms aspecific immune complex with the protein.

Preferred epitopes encompassed by the antigenic peptide are regions thatare located on the surface of the protein, e.g., hydrophilic regions.FIGS. 2, 7, and 10 are hydropathy plots of the proteins of theinvention. These plots or similar analyses can be used to identifyhydrophilic regions.

An immunogen typically is used to prepare antibodies by immunizing asuitable subject, (e.g., rabbit, goat, mouse or other mammal). Anappropriate immunogenic preparation can contain, for example,recombinantly expressed or chemically synthesized polypeptide. Thepreparation can further include an adjuvant, such as Freund's completeor incomplete adjuvant, or similar immunostimulatory agent.

Accordingly, another aspect of the invention pertains to antibodiesdirected against a polypeptide of the invention. The term “antibody” asused herein refers to immunoglobulin molecules and immunologicallyactive portions of immunoglobulin molecules, i.e., molecules thatcontain an antigen binding site which specifically binds an antigen,such as a polypeptide of the invention. A molecule which specificallybinds to a given polypeptide of the invention is a molecule which bindsthe polypeptide, but does not substantially bind other molecules in asample, e.g., a biological sample, which naturally contains thepolypeptide. Examples of immunologically active portions ofimmunoglobulin molecules include F(ab) and F(ab′)₂ fragments which canbe generated by treating the antibody with an enzyme such as pepsin. Theinvention provides polyclonal and monoclonal antibodies. The term“monoclonal antibody” or “monoclonal antibody composition”, as usedherein, refers to a population of antibody molecules that contain onlyone species of an antigen binding site capable of immunoreacting with aparticular epitope.

Polyclonal antibodies can be prepared as described above by immunizing asuitable subject with a polypeptide of the invention as an immunogen.The antibody titer in the immunized subject can be monitored over timeby standard techniques, such as with an enzyme linked immunosorbentassay (ELISA) using immobilized polypeptide. If desired, the antibodymolecules can be isolated from the mammal (e.g., from the blood) andfurther purified by well-known techniques, such as protein Achromatography to obtain the IgG fraction. At an appropriate time afterimmunization, e.g., when the specific antibody titers are highest,antibody-producing cells can be obtained from the subject and used toprepare monoclonal antibodies by standard techniques, such as thehybridoma technique originally described by Kohler and Milstein (1975)Nature 256:495-497, the human B cell hybridoma technique (Kozbor et al.(1983) Immunol. Today 4:72), the EBV-hybridoma technique (Cole et al.(1985), Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.,pp. 77-96) or trioma techniques. The technology for producing hybridomasis well known (see generally Current Protocols in Immunology (1994)Coligan et al. (eds.) John Wiley & Sons, Inc., New York, N.Y.).Hybridoma cells producing a monoclonal antibody of the invention aredetected by screening the hybridoma culture supernatants for antibodiesthat bind the polypeptide of interest, e.g., using a standard ELISAassay.

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal antibody directed against a polypeptide of the invention canbe identified and isolated by screening a recombinant combinatorialimmunoglobulin library (e.g., an antibody phage display library) withthe polypeptide of interest. Kits for generating and screening phagedisplay libraries are commercially available (e.g., the PharmaciaRecombinant Phage Antibody System, Catalog No. 27-9400-01; and theStratagene SurfZAP™ Phage Display Kit, Catalog No. 240612).Additionally, examples of methods and reagents particularly amenable foruse in generating and screening antibody display library can be foundin, for example, U.S. Pat. No. 5,223,409; PCT Publication No. WO92/18619; PCT Publication No. WO 91/17271; PCT Publication No. WO92/20791; PCT Publication No. WO 92/15679; PCT Publication No. WO93/01288; PCT Publication No. WO 92/01047; PCT Publication No. WO92/09690; PCT Publication No. WO 90/02809; Fuchs et al. (1991)Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al.(1993) EMBO J. 12:725-734.

Additionally, recombinant antibodies, such as chimeric and humanizedmonoclonal antibodies, comprising both human and non-human portions,which can be made using standard recombinant DNA techniques, are withinthe scope of the invention. Such chimeric and humanized monoclonalantibodies can be produced by recombinant DNA techniques known in theart, for example using methods described in PCT Publication No. WO87/02671; European Patent Application 184,187; European PatentApplication 171,496; European Patent Application 173,494; PCTPublication No. WO 86/01533; U.S. Pat. No. 4,816,567; European PatentApplication 125,023; Better et al. (1988) Science 240:1041-1043; Liu etal. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J.Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA84:214-218; Nishimura et al. (1987) Canc. Res. 47:999-1005; Wood et al.(1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst.80:1553-1559); Morrison (1985) Science 229:1202-1207; Oi et al. (1986)Bio/Techniques 4:214; U.S. Pat. No. 5,225,539; Jones et al. (1986)Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; andBeidler et al. (1988) J. Immunol. 141:4053-4060.

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Such antibodies can be produced usingtransgenic mice which are incapable of expressing endogenousimmunoglobulin heavy and light chains genes, but which can express humanheavy and light chain genes. The transgenic mice are immunized in thenormal fashion with a selected antigen, e.g., all or a portion of apolypeptide of the invention. Monoclonal antibodies directed against theantigen can be obtained using conventional hybridoma technology. Thehuman immunoglobulin transgenes harbored by the transgenic micerearrange during B cell differentiation, and subsequently undergo classswitching and somatic mutation. Thus, using such a technique, it ispossible to produce therapeutically useful IgG, IgA and IgE antibodies.For an overview of this technology for producing human antibodies, seeLonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93). For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g., U.S. Pat. No. 5,625,126; U.S. Pat. No. 5,633,425; U.S. Pat. No.5,569,825; U.S. Pat. No. 5,661,016; and U.S. Pat. No. 5,545,806. Inaddition, companies such as Abgenix, Inc. (Freemont, Calif.), can beengaged to provide human antibodies directed against a selected antigenusing technology similar to that described above.

Completely human antibodies which recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a murineantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al. (1994) Bio/technology12:899-903).

An antibody directed against a polypeptide of the invention (e.g.,monoclonal antibody) can be used to isolate the polypeptide by standardtechniques, such as affinity chromatography or immunoprecipitation.Moreover, such an antibody can be used to detect the protein (e.g., in acellular lysate or cell supernatant) in order to evaluate the abundanceand pattern of expression of the polypeptide. The antibodies can also beused diagnostically to monitor protein levels in tissue as part of aclinical testing procedure, e.g., to, for example, determine theefficacy of a given treatment regimen. Detection can be facilitated bycoupling the antibody to a detectable substance. Examples of detectablesubstances include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials, bioluminescent materials, andradioactive materials. Examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

III. Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding a polypeptide ofthe invention (or a portion thereof). As used herein, the term “vector”refers to a nucleic acid molecule capable of transporting anothernucleic acid to which it has been linked. One type of vector is a“plasmid”, which refers to a circular double stranded DNA loop intowhich additional DNA segments can be ligated. Another type of vector isa viral vector, wherein additional DNA segments can be ligated into theviral genome. Certain vectors are capable of autonomous replication in ahost cell into which they are introduced (e.g., bacterial vectors havinga bacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome. Moreover, certain vectors,expression vectors, are capable of directing the expression of genes towhich they are operably linked. In general, expression vectors ofutility in recombinant DNA techniques are often in the form of plasmids(vectors). However, the invention is intended to include such otherforms of expression vectors, such as viral vectors (e.g., replicationdefective retroviruses, adenoviruses and adeno-associated viruses),which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell. This means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, which is operably linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerwhich allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term “regulatory sequence” isintended to include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel, Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatorysequences include those which direct constitutive expression of anucleotide sequence in many types of host cell and those which directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the host cell to be transformed, thelevel of expression of protein desired, etc. The expression vectors ofthe invention can be introduced into host cells to thereby produceproteins or peptides, including fusion proteins or peptides, encoded bynucleic acids as described herein.

The recombinant expression vectors of the invention can be designed forexpression of a polypeptide of the invention in prokaryotic (e.g., E.coli) or eukaryotic cells (e.g., insect cells (using baculovirusexpression vectors), yeast cells or mammalian cells). Suitable hostcells are discussed further in Goeddel, supra. Alternatively, therecombinant expression vector can be transcribed and translated invitro, for example using T7 promoter regulatory sequences and T7polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith and Johnson (1988) Gene 67:31-40), pMAL (New England Biolabs,Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuseglutathione S-transferase (GST), maltose E binding protein, or proteinA, respectively, to the target recombinant protein.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al., (1988) Gene 69:301-315) and pET 11d (Studieret al., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 60-89). Target gene expression from thepTrc vector relies on host RNA polymerase transcription from a hybridtrp-lac fusion promoter. Target gene expression from the pET 11d vectorrelies on transcription from a T7 gn10-lac fusion promoter mediated by acoexpressed viral RNA polymerase (T7 gn1). This viral polymerase issupplied by host strains BL21(DE3) or HMS174(DE3) from a resident λprophage harboring a T7 gn1 gene under the transcriptional control ofthe lacUV 5 promoter.

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman, GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 119-128). Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in E. coli (Wada et al. (1992) Nucleic AcidsRes. 20:2111-2118). Such alteration of nucleic acid sequences of theinvention can be carried out by standard DNA synthesis techniques.

In another embodiment, the expression vector is a yeast expressionvector. Examples of vectors for expression in yeast S. cerivisae includepYepSec1 (Baldari et al. (1987) EMBO J. 6:229-234), pMFa (Kurjan andHerskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al. (1987) Gene54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), andpPicZ (Invitrogen Corp, San Diego, Calif.).

Alternatively, the expression vector is a baculovirus expression vector.Baculovirus vectors available for expression of proteins in culturedinsect cells (e.g., Sf 9 cells) include the pAc series (Smith et al.(1983) Mol. Cell. Biol. 3:2156-2165) and the pVL series (Lucklow andSummers (1989) Virology 170:31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed (1987) Nature 329:840)and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195). When used inmammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook etal., supra.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.(1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame andEaton (1988) Adv. Immunol. 43:235-275), in particular promoters of Tcell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) andimmunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen andBaltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci.USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985)Science 230:912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, for example the murine hox promoters (Kessel and Gruss(1990) Science 249:374-379) and the beta-fetoprotein promoter (Campesand Tilghman (1989) Genes Dev. 3:537-546).

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperably linked to a regulatory sequence in a manner which allows forexpression (by transcription of the DNA molecule) of an RNA moleculewhich is antisense to the mRNA encoding a polypeptide of the invention.Regulatory sequences operably linked to a nucleic acid cloned in theantisense orientation can be chosen which direct the continuousexpression of the antisense RNA molecule in a variety of cell types, forinstance viral promoters and/or enhancers, or regulatory sequences canbe chosen which direct constitutive, tissue specific or cell typespecific expression of antisense RNA. The antisense expression vectorcan be in the form of a recombinant plasmid, phagemid or attenuatedvirus in which antisense nucleic acids are produced under the control ofa high efficiency regulatory region, the activity of which can bedetermined by the cell type into which the vector is introduced. For adiscussion of the regulation of gene expression using antisense genessee Weintraub et al. (Reviews—Trends in Genetics, Vol. 1(1) 1986).

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but to the progeny or potential progeny of sucha cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A host cell can be any prokaryotic (e.g., E. coli) or eukaryotic cell(e.g., insect cells, yeast or mammalian cells).

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid into a host cell, including calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection, orelectroporation. Suitable methods for transforming or transfecting hostcells can be found in Sambrook, et al. (supra), and other laboratorymanuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., for resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin and methotrexate. Cells stablytransfected with the introduced nucleic acid can be identified by drugselection (e.g., cells that have incorporated the selectable marker genewill survive, while the other cells die).

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce a polypeptide of the invention.Accordingly, the invention further provides methods for producing apolypeptide of the invention using the host cells of the invention. Inone embodiment, the method comprises culturing the host cell ofinvention (into which a recombinant expression vector encoding apolypeptide of the invention has been introduced) in a suitable mediumsuch that the polypeptide is produced. In another embodiment, the methodfurther comprises isolating the polypeptide from the medium or the hostcell.

The host cells of the invention can also be used to produce nonhumantransgenic animals. For example, in one embodiment, a host cell of theinvention is a fertilized oocyte or an embryonic stem cell into which asequence encoding a polypeptide of the invention has been introduced.Such host cells can then be used to create non-human transgenic animalsin which exogenous sequences encoding a polypeptide of the inventionhave been introduced into their genome or homologous recombinant animalsin which endogenous encoding a polypeptide of the invention sequenceshave been altered. Such animals are useful for studying the functionand/or activity of the polypeptide and for identifying and/or evaluatingmodulators of polypeptide activity. As used herein, a “transgenicanimal” is a non-human animal, preferably a mammal, more preferably arodent such as a rat or mouse, in which one or more of the cells of theanimal includes a transgene. Other examples of transgenic animalsinclude non-human primates, sheep, dogs, cows, goats, chickens,amphibians, etc. A transgene is exogenous DNA which is integrated intothe genome of a cell from which a transgenic animal develops and whichremains in the genome of the mature animal, thereby directing theexpression of an encoded gene product in one or more cell types ortissues of the transgenic animal. As used herein, an “homologousrecombinant animal” is a non-human animal, preferably a mammal, morepreferably a mouse, in which an endogenous gene has been altered byhomologous recombination between the endogenous gene and an exogenousDNA molecule introduced into a cell of the animal, e.g., an embryoniccell of the animal, prior to development of the animal.

A transgenic animal of the invention can be created by introducingnucleic acid encoding a polypeptide of the invention (or a homologuethereof) into the male pronuclei of a fertilized oocyte, e.g., bymicroinjection, retroviral infection, and allowing the oocyte to developin a pseudopregnant female foster animal. Intronic sequences andpolyadenylation signals can also be included in the transgene toincrease the efficiency of expression of the transgene. Atissue-specific regulatory sequence(s) can be operably linked to thetransgene to direct expression of the polypeptide of the invention toparticular cells. Methods for generating transgenic animals via embryomanipulation and microinjection, particularly animals such as mice, havebecome conventional in the art and are described, for example, in U.S.Pat. Nos. 4,736,866 and 4,870,009, U.S. Pat. No. 4,873,191 and in Hogan,Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1986). Similar methods are used for productionof other transgenic animals. A transgenic founder animal can beidentified based upon the presence of the transgene in its genome and/orexpression of mRNA encoding the transgene in tissues or cells of theanimals. A transgenic founder animal can then be used to breedadditional animals carrying the transgene. Moreover, transgenic animalscarrying the transgene can further be bred to other transgenic animalscarrying other transgenes.

To create an homologous recombinant animal, a vector is prepared whichcontains at least a portion of a gene encoding a polypeptide of theinvention into which a deletion, addition or substitution has beenintroduced to thereby alter, e.g., functionally disrupt, the gene. In apreferred embodiment, the vector is designed such that, upon homologousrecombination, the endogenous gene is functionally disrupted (i.e., nolonger encodes a functional protein; also referred to as a “knock out”vector). Alternatively, the vector can be designed such that, uponhomologous recombination, the endogenous gene is mutated or otherwisealtered but still encodes functional protein (e.g., the upstreamregulatory region can be altered to thereby alter the expression of theendogenous protein). In the homologous recombination vector, the alteredportion of the gene is flanked at its 5′ and 3′ ends by additionalnucleic acid of the gene to allow for homologous recombination to occurbetween the exogenous gene carried by the vector and an endogenous genein an embryonic stem cell. The additional flanking nucleic acidsequences are of sufficient length for successful homologousrecombination with the endogenous gene. Typically, several kilobases offlanking DNA (both at the 5′ and 3′ ends) are included in the vector(see, e.g., Thomas and Capecchi (1987) Cell 51:503 for a description ofhomologous recombination vectors). The vector is introduced into anembryonic stem cell line (e.g., by electroporation) and cells in whichthe introduced gene has homologously recombined with the endogenous geneare selected (see, e.g., Li et al. (1992) Cell 69:915). The selectedcells are then injected into a blastocyst of an animal (e.g., a mouse)to form aggregation chimeras (see, e.g., Bradley in Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, Robertson, ed. (IRL, Oxford,1987) pp. 113-152). A chimeric embryo can then be implanted into asuitable pseudopregnant female foster animal and the embryo brought toterm. Progeny harboring the homologously recombined DNA in their germcells can be used to breed animals in which all cells of the animalcontain the homologously recombined DNA by germline transmission of thetransgene. Methods for constructing homologous recombination vectors andhomologous recombinant animals are described further in Bradley (1991)Current Opinion in Bio/Technology 2:823-829 and in PCT Publication Nos.WO 90/11354, WO 91/01140, WO 92/0968, and WO 93/04169.

In another embodiment, transgenic non-human animals can be producedwhich contain selected systems which allow for regulated expression ofthe transgene. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al. (1992) Proc. Natl. Acad.Sci. USA 89:6232-6236. Another example of a recombinase system is theFLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al.(1991) Science 251:1351-1355. If a cre/loxP recombinase system is usedto regulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein are required.Such animals can be provided through the construction of “double”transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in Wilmut et al. (1997)Nature 385:810-813 and PCT Publication NOS. WO 97/07668 and WO 97/07669.

IV. Pharmaceutical Compositions

The nucleic acid molecules, polypeptides, and antibodies (also referredto herein as “active compounds”) of the invention can be incorporatedinto pharmaceutical compositions suitable for administration. Suchcompositions typically comprise the nucleic acid molecule, protein, orantibody and a pharmaceutically acceptable carrier. As used herein thelanguage “pharmaceutically acceptable carrier” is intended to includeany and all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active compound, use thereof in the compositionsis contemplated. Supplementary active compounds can also be incorporatedinto the compositions.

The invention includes methods for preparing pharmaceutical compositionsfor modulating the expression or activity of a polypeptide or nucleicacid of the invention. Such methods comprise formulating apharmaceutically acceptable carrier with an agent which modulatesexpression or activity of a polypeptide or nucleic acid of theinvention. Such compositions can further include additional activeagents. Thus, the invention further includes methods for preparing apharmaceutical composition by formulating a pharmaceutically acceptablecarrier with an agent which modulates expression or activity of apolypeptide or nucleic acid of the invention and one or more additionalactive compounds.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF; Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., a polypeptide or antibody) in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle which contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingwhich yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed.

Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from a pressurized container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

For antibodies, the preferred dosage is 0.1 mg/kg to 100 mg/kg of bodyweight (generally 10 mg/kg to 20 mg/kg). If the antibody is to act inthe brain, a dosage of 50 mg/kg to 100 mg/kg is usually appropriate.Generally, partially human antibodies and fully human antibodies have alonger half-life within the human body than other antibodies.Accordingly, lower dosages and less frequent administration is oftenpossible. Modifications such as lipidation can be used to stabilizeantibodies and to enhance uptake and tissue penetration (e.g., into thebrain). A method for lipidation of antibodies is described by Cruikshanket al. ((1997) J. Acquired Immune Deficiency Syndromes and HumanRetrovirology 14:193).

The nucleic acid molecules of the invention can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by, for example, intravenous injection, localadministration (U.S. Pat. No. 5,328,470) or by stereotactic injection(see, e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057).The pharmaceutical preparation of the gene therapy vector can includethe gene therapy vector in an acceptable diluent, or can comprise a slowrelease matrix in which the gene delivery vehicle is imbedded.Alternatively, where the complete gene delivery vector can be producedintact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

V. Uses and Methods of the Invention

The nucleic acid molecules, proteins, protein homologues, and antibodiesdescribed herein can be used in one or more of the following methods: a)screening assays; b) detection assays (e.g., chromosomal mapping, tissuetyping, forensic biology); c) predictive medicine (e.g., diagnosticassays, prognostic assays, monitoring clinical trials, andpharmacogenomics); and d) methods of treatment (e.g., therapeutic andprophylactic). The isolated nucleic acid molecules of the invention canbe used to express proteins (e.g., via a recombinant expression vectorin a host cell in gene therapy applications), to detect mRNA (e.g., in abiological sample) or a genetic lesion, and to modulate activity of apolypeptide of the invention. In addition, the polypeptides of theinvention can be used to screen drugs or compounds which modulateactivity or expression of a polypeptide of the invention as well as totreat disorders characterized by insufficient or excessive production ofa protein of the invention or production of a form of a protein of theinvention which has decreased or aberrant activity compared to the wildtype protein. In addition, the antibodies of the invention can be usedto detect and isolate a protein of the and modulate activity of aprotein of the invention.

This invention further pertains to novel agents identified by theabove-described screening assays and uses thereof for treatments asdescribed herein.

A. Screening Assays

The invention provides a method (also referred to herein as a “screeningassay”) for identifying modulators, i.e., candidate or test compounds oragents (e.g., peptides, peptidomimetics, small molecules or other drugs)which bind to polypeptide of the invention or have a stimulatory orinhibitory effect on, for example, expression or activity of apolypeptide of the invention.

In one embodiment, the invention provides assays for screening candidateor test compounds which bind to or modulate the activity of themembrane-bound form of a polypeptide of the invention or biologicallyactive portion thereof. The test compounds of the present invention canbe obtained using any of the numerous approaches in combinatoriallibrary methods known in the art, including: biological libraries;spatially addressable parallel solid phase or solution phase libraries;synthetic library methods requiring deconvolution; the “one-beadone-compound” library method; and synthetic library methods usingaffinity chromatography selection. The biological library approach islimited to peptide libraries, while the other four approaches areapplicable to peptide, non-peptide oligomer or small molecule librariesof compounds (Lam (1997) Anticancer Drug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422;Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al. (1993)Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl.33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; andGallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Bio/Techniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (U.S. Pat.No. 5,223,409), spores (U.S. Pat. Nos. 5,571,698; 5,403,484; and5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA89:1865-1869) or phage (Scott and Smith (1990) Science 249:386-390;Devlin (1990) Science 249:404-406; Cwirla et al. (1990) Proc. Natl.Acad. Sci. USA 87:6378-6382; and Felici (1991) J. Mol. Biol.222:301-310).

In one embodiment, an assay is a cell-based assay in which a cell whichexpresses a membrane-bound form of a polypeptide of the invention, or abiologically active portion thereof, on the cell surface is contactedwith a test compound and the ability of the test compound to bind to thepolypeptide determined. The cell, for example, can be a yeast cell or acell of mammalian origin. Determining the ability of the test compoundto bind to the polypeptide can be accomplished, for example, by couplingthe test compound with a radioisotope or enzymatic label such thatbinding of the test compound to the polypeptide or biologically activeportion thereof can be determined by detecting the labeled compound in acomplex. For example, test compounds can be labeled with ¹²⁵I, ³⁵S, ¹⁴C,or ³H, either directly or indirectly, and the radioisotope detected bydirect counting of radioemmission or by scintillation counting.Alternatively, test compounds can be enzymatically labeled with, forexample, horseradish peroxidase, alkaline phosphatase, or luciferase,and the enzymatic label detected by determination of conversion of anappropriate substrate to product. In a preferred embodiment, the assaycomprises contacting a cell which expresses a membrane-bound form of apolypeptide of the invention, or a biologically active portion thereof,on the cell surface with a known compound which binds the polypeptide toform an assay mixture, contacting the assay mixture with a testcompound, and determining the ability of the test compound to interactwith the polypeptide, wherein determining the ability of the testcompound to interact with the polypeptide comprises determining theability of the test compound to preferentially bind to the polypeptideor a biologically active portion thereof as compared to the knowncompound.

In another embodiment, an assay is a cell-based assay comprisingcontacting a cell expressing a membrane-bound form of a polypeptide ofthe invention, or a biologically active portion thereof, on the cellsurface with a test compound and determining the ability of the testcompound to modulate (e.g., stimulate or inhibit) the activity of thepolypeptide or biologically active portion thereof. Determining theability of the test compound to modulate the activity of the polypeptideor a biologically active portion thereof can be accomplished, forexample, by determining the ability of the polypeptide protein to bindto or interact with a target molecule.

Determining the ability of a polypeptide of the invention to bind to orinteract with a target molecule can be accomplished by one of themethods described above for determining direct binding. As used herein,a “target molecule” is a molecule with which a selected polypeptide(e.g., a polypeptide of the invention) binds or interacts with innature, for example, a molecule on the surface of a cell which expressesthe selected protein, a molecule on the surface of a second cell, amolecule in the extracellular milieu, a molecule associated with theinternal surface of a cell membrane or a cytoplasmic molecule. A targetmolecule can be a polypeptide of the invention or some other polypeptideor protein. For example, a target molecule can be a component of asignal transduction pathway which facilitates transduction of anextracellular signal (e.g., a signal generated by binding of a compoundto a polypeptide of the invention) through the cell membrane and intothe cell or a second intercellular protein which has catalytic activityor a protein which facilitates the association of downstream signalingmolecules with a polypeptide of the invention. Determining the abilityof a polypeptide of the invention to bind to or interact with a targetmolecule can be accomplished by determining the activity of the targetmolecule. For example, the activity of the target molecule can bedetermined by detecting induction of a cellular second messenger of thetarget (e.g., intracellular Ca²⁺, diacylglycerol, IP3, etc.), detectingcatalytic/enzymatic activity of the target on an appropriate substrate,detecting the induction of a reporter gene (e.g., a regulatory elementthat is responsive to a polypeptide of the invention operably linked toa nucleic acid encoding a detectable marker, e.g., luciferase), ordetecting a cellular response, for example, cellular differentiation, orcell proliferation.

In yet another embodiment, an assay of the present invention is acell-free assay comprising contacting a polypeptide of the invention orbiologically active portion thereof with a test compound and determiningthe ability of the test compound to bind to the polypeptide orbiologically active portion thereof. Binding of the test compound to thepolypeptide can be determined either directly or indirectly as describedabove. In a preferred embodiment, the assay includes contacting thepolypeptide of the invention or biologically active portion thereof witha known compound which binds the polypeptide to form an assay mixture,contacting the assay mixture with a test compound, and determining theability of the test compound to interact with the polypeptide, whereindetermining the ability of the test compound to interact with thepolypeptide comprises determining the ability of the test compound topreferentially bind to the polypeptide or biologically active portionthereof as compared to the known compound.

In another embodiment, an assay is a cell-free assay comprisingcontacting a polypeptide of the invention or biologically active portionthereof with a test compound and determining the ability of the testcompound to modulate (e.g., stimulate or inhibit) the activity of thepolypeptide or biologically active portion thereof. Determining theability of the test compound to modulate the activity of the polypeptidecan be accomplished, for example, by determining the ability of thepolypeptide to bind to a target molecule by one of the methods describedabove for determining direct binding. In an alternative embodiment,determining the ability of the test compound to modulate the activity ofthe polypeptide can be accomplished by determining the ability of thepolypeptide of the invention to further modulate the target molecule.For example, the catalytic/enzymatic activity of the target molecule onan appropriate substrate can be determined as previously described.

In yet another embodiment, the cell-free assay comprises contacting apolypeptide of the invention or biologically active portion thereof witha known compound which binds the polypeptide to form an assay mixture,contacting the assay mixture with a test compound, and determining theability of the test compound to interact with the polypeptide, whereindetermining the ability of the test compound to interact with thepolypeptide comprises determining the ability of the polypeptide topreferentially bind to or modulate the activity of a target molecule.

The cell-free assays of the present invention are amenable to use ofboth a soluble form or the membrane-bound form of a polypeptide of theinvention. In the case of cell-free assays comprising the membrane-boundform of the polypeptide, it may be desirable to utilize a solubilizingagent such that the membrane-bound form of the polypeptide is maintainedin solution. Examples of such solubilizing agents include non-ionicdetergents such as n-octylglucoside, n-dodecylglucoside,n-octylmaltoside, octanoyl-N-methylglucamide,decanoyl-N-methylglucamide, Triton X-100, Triton X-114, Thesit,Isotridecypoly(ethylene glycol ether)n,3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS),3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate(CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane sulfonate.

In more than one embodiment of the above assay methods of the presentinvention, it may be desirable to immobilize either the polypeptide ofthe invention or its target molecule to facilitate separation ofcomplexed from uncomplexed forms of one or both of the proteins, as wellas to accommodate automation of the assay. Binding of a test compound tothe polypeptide, or interaction of the polypeptide with a targetmolecule in the presence and absence of a candidate compound, can beaccomplished in any vessel suitable for containing the reactants.Examples of such vessels include microtitre plates, test tubes, andmicro-centrifuge tubes. In one embodiment, a fusion protein can beprovided which adds a domain that allows one or both of the proteins tobe bound to a matrix. For example, glutathione-S-transferase fusionproteins or glutathione-S-transferase fusion proteins can be adsorbedonto glutathione sepharose beads (Sigma Chemical; St. Louis, Mo.) orglutathione derivatized microtitre plates, which are then combined withthe test compound or the test compound and either the non-adsorbedtarget protein or A polypeptide of the invention, and the mixtureincubated under conditions conducive to complex formation (e.g., atphysiological conditions for salt and pH). Following incubation, thebeads or microtitre plate wells are washed to remove any unboundcomponents and complex formation is measured either directly orindirectly, for example, as described above. Alternatively, thecomplexes can be dissociated from the matrix, and the level of bindingor activity of the polypeptide of the invention can be determined usingstandard techniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, either thepolypeptide of the invention or its target molecule can be immobilizedutilizing conjugation of biotin and streptavidin. Biotinylatedpolypeptide of the invention or target molecules can be prepared frombiotin-NHS (N-hydroxy-succinimide) using techniques well known in theart (e.g., biotinylation kit, Pierce Chemicals; Rockford, Ill.), andimmobilized in the wells of streptavidin-coated 96 well plates (PierceChemical). Alternatively, antibodies reactive with the polypeptide ofthe invention or target molecules but which do not interfere withbinding of the polypeptide of the invention to its target molecule canbe derivatized to the wells of the plate, and unbound target orpolypeptide of the invention trapped in the wells by antibodyconjugation. Methods for detecting such complexes, in addition to thosedescribed above for the GST-immobilized complexes, includeimmunodetection of complexes using antibodies reactive with thepolypeptide of the invention or target molecule, as well asenzyme-linked assays which rely on detecting an enzymatic activityassociated with the polypeptide of the invention or target molecule.

In another embodiment, modulators of expression of a polypeptide of theinvention are identified in a method in which a cell is contacted with acandidate compound and the expression of the selected mRNA or protein(i.e., the mRNA or protein corresponding to a polypeptide or nucleicacid of the invention) in the cell is determined. The level ofexpression of the selected mRNA or protein in the presence of thecandidate compound is compared to the level of expression of theselected mRNA or protein in the absence of the candidate compound. Thecandidate compound can then be identified as a modulator of expressionof the polypeptide of the invention based on this comparison. Forexample, when expression of the selected mRNA or protein is greater(statistically significantly greater) in the presence of the candidatecompound than in its absence, the candidate compound is identified as astimulator of the selected mRNA or protein expression. Alternatively,when expression of the selected mRNA or protein is less (statisticallysignificantly less) in the presence of the candidate compound than inits absence, the candidate compound is identified as an inhibitor of theselected mRNA or protein expression. The level of the selected mRNA orprotein expression in the cells can be determined by methods describedherein.

In yet another aspect of the invention, a polypeptide of the inventionscan be used as “bait proteins” in a two-hybrid assay or three hybridassay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartelet al. (1993) Bio/Techniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and PCT Publication No. WO 94/10300), to identify otherproteins, which bind to or interact with the polypeptide of theinvention and modulate activity of the polypeptide of the invention.Such binding proteins are also likely to be involved in the propagationof signals by the polypeptide of the inventions as, for example,upstream or downstream elements of a signaling pathway involving thepolypeptide of the invention.

This invention further pertains to novel agents identified by theabove-described screening assays and uses thereof for treatments asdescribed herein.

B. Detection Assays

Portions or fragments of the cDNA sequences identified herein (and thecorresponding complete gene sequences) can be used in numerous ways aspolynucleotide reagents. For example, these sequences can be used to:(i) map their respective genes on a chromosome and, thus, locate generegions associated with genetic disease; (ii) identify an individualfrom a minute biological sample (tissue typing); and (iii) aid inforensic identification of a biological sample. These applications aredescribed in the subsections below.

1. Chromosome Mapping

Once the sequence (or a portion of the sequence) of a gene has beenisolated, this sequence can be used to map the location of the gene on achromosome. Accordingly, nucleic acid molecules described herein orfragments thereof, can be used to map the location of the correspondinggenes on a chromosome. The mapping of the sequences to chromosomes is animportant first step in correlating these sequences with genesassociated with disease.

Briefly, genes can be mapped to chromosomes by preparing PCR primers(preferably 15-25 bp in length) from the sequence of a gene of theinvention. Computer analysis of the sequence of a gene of the inventioncan be used to rapidly select primers that do not span more than oneexon in the genomic DNA, thus complicating the amplification process.These primers can then be used for PCR screening of somatic cell hybridscontaining individual human chromosomes. Only those hybrids containingthe human gene corresponding to the gene sequences will yield anamplified fragment. For a review of this technique, see D'Eustachio etal. ((1983) Science 220:919-924).

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular sequence to a particular chromosome. Three or more sequencescan be assigned per day using a single thermal cycler. Using the nucleicacid sequences of the invention to design oligonucleotide primers,sublocalization can be achieved with panels of fragments from specificchromosomes. Other mapping strategies which can similarly be used to mapa gene to its chromosome include in situ hybridization (described in Fanet al. (1990) Proc. Natl. Acad. Sci. USA 87:6223-27), pre-screening withlabeled flow-sorted chromosomes (CITE), and pre-selection byhybridization to chromosome specific cDNA libraries. Fluorescence insitu hybridization (FISH) of a DNA sequence to a metaphase chromosomalspread can further be used to provide a precise chromosomal location inone step. For a review of this technique, see Verma et al., (HumanChromosomes: A Manual of Basic Techniques (Pergamon Press, New York,1988)).

Reagents for chromosome mapping can be used individually to mark asingle chromosome or a single site on that chromosome, or panels ofreagents can be used for marking multiple sites and/or multiplechromosomes. Reagents corresponding to noncoding regions of the genesactually are preferred for mapping purposes. Coding sequences are morelikely to be conserved within gene families, thus increasing the chanceof cross hybridizations during chromosomal mapping.

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. (Such data are found, for example, in V.McKusick, Mendelian Inheritance in Man, available on-line through JohnsHopkins University Welch Medical Library). The relationship betweengenes and disease, mapped to the same chromosomal region, can then beidentified through linkage analysis (co-inheritance of physicallyadjacent genes), described in, e.g., Egeland et al. (1987) Nature325:783-787.

Moreover, differences in the DNA sequences between individuals affectedand unaffected with a disease associated with a gene of the inventioncan be determined. If a mutation is observed in some or all of theaffected individuals but not in any unaffected individuals, then themutation is likely to be the causative agent of the particular disease.Comparison of affected and unaffected individuals generally involvesfirst looking for structural alterations in the chromosomes such asdeletions or translocations that are visible from chromosome spreads ordetectable using PCR based on that DNA sequence. Ultimately, completesequencing of genes from several individuals can be performed to confirmthe presence of a mutation and to distinguish mutations frompolymorphisms.

2. Tissue Typing

The nucleic acid sequences of the present invention can also be used toidentify individuals from minute biological samples. The United Statesmilitary, for example, is considering the use of restriction fragmentlength polymorphism (RFLP) for identification of its personnel. In thistechnique, an individual's genomic DNA is digested with one or morerestriction enzymes, and probed on a Southern blot to yield unique bandsfor identification. This method does not suffer from the currentlimitations of “Dog Tags” which can be lost, switched, or stolen, makingpositive identification difficult. The sequences of the presentinvention are useful as additional DNA markers for RFLP (described inU.S. Pat. No. 5,272,057).

Furthermore, the sequences of the present invention can be used toprovide an alternative technique which determines the actualbase-by-base DNA sequence of selected portions of an individual'sgenome. Thus, the nucleic acid sequences described herein can be used toprepare two PCR primers from the 5′ and 3′ ends of the sequences. Theseprimers can then be used to amplify an individual's DNA and subsequentlysequence it.

Panels of corresponding DNA sequences from individuals, prepared in thismanner, can provide unique individual identifications, as eachindividual will have a unique set of such DNA sequences due to allelicdifferences. The sequences of the present invention can be used toobtain such identification sequences from individuals and from tissue.The nucleic acid sequences of the invention uniquely represent portionsof the human genome. Allelic variation occurs to some degree in thecoding regions of these sequences, and to a greater degree in thenoncoding regions. It is estimated that allelic variation betweenindividual humans occurs with a frequency at about once per each 500bases. Each of the sequences described herein can, to some degree, beused as a standard against which DNA from an individual can be comparedfor identification purposes. Because greater numbers of polymorphismsoccur in the noncoding regions, fewer sequences are necessary todifferentiate individuals. The noncoding sequences of SEQ ID NO:1, 13,or 19 can comfortably provide positive individual identification with apanel of perhaps 10 to 1,000 primers which each yield a noncodingamplified sequence of 100 bases. If predicted coding sequences, such asthose in SEQ ID NO:2, 14, 20, or 44 are used, a more appropriate numberof primers for positive individual identification would be 500-2,000.

If a panel of reagents from the nucleic acid sequences described hereinis used to generate a unique identification database for an individual,those same reagents can later be used to identify tissue from thatindividual. Using the unique identification database, positiveidentification of the individual, living or dead, can be made fromextremely small tissue samples.

3. Use of Partial Gene Sequences in Forensic Biology

DNA-based identification techniques can also be used in forensicbiology. Forensic biology is a scientific field employing genetic typingof biological evidence found at a crime scene as a means for positivelyidentifying, for example, a perpetrator of a crime. To make such anidentification, PCR technology can be used to amplify DNA sequencestaken from very small biological samples such as tissues, e.g., hair orskin, or body fluids, e.g., blood, saliva, or semen found at a crimescene. The amplified sequence can then be compared to a standard,thereby allowing identification of the origin of the biological sample.

The sequences of the present invention can be used to providepolynucleotide reagents, e.g., PCR primers, targeted to specific loci inthe human genome, which can enhance the reliability of DNA-basedforensic identifications by, for example, providing another“identification marker” (i.e. another DNA sequence that is unique to aparticular individual). As mentioned above, actual base sequenceinformation can be used for identification as an accurate alternative topatterns formed by restriction enzyme generated fragments. Sequencestargeted to noncoding regions are particularly appropriate for this useas greater numbers of polymorphisms occur in the noncoding regions,making it easier to differentiate individuals using this technique.Examples of polynucleotide reagents include the nucleic acid sequencesof the invention or portions thereof, e.g., fragments derived fromnoncoding regions having a length of at least 20 or 30 bases.

The nucleic acid sequences described herein can further be used toprovide polynucleotide reagents, e.g., labeled or labelable probes whichcan be used in, for example, an in situ hybridization technique, toidentify a specific tissue, e.g., brain tissue. This can be very usefulin cases where a forensic pathologist is presented with a tissue ofunknown origin. Panels of such probes can be used to identify tissue byspecies and/or by organ type.

C. Predictive Medicine

The present invention also pertains to the field of predictive medicinein which diagnostic assays, prognostic assays, pharmacogenomics, andmonitoring clinical trails are used for prognostic (predictive) purposesto thereby treat an individual prophylactically. Accordingly, one aspectof the present invention relates to diagnostic assays for determiningexpression of a polypeptide or nucleic acid of the invention and/oractivity of a polypeptide of the invention, in the context of abiological sample (e.g., blood, serum, cells, tissue) to therebydetermine whether an individual is afflicted with a disease or disorder,or is at risk of developing a disorder, associated with aberrantexpression or activity of a polypeptide of the invention. The inventionalso provides for prognostic (or predictive) assays for determiningwhether an individual is at risk of developing a disorder associatedwith aberrant expression or activity of a polypeptide of the invention.For example, mutations in a gene of the invention can be assayed in abiological sample. Such assays can be used for prognostic or predictivepurpose to thereby prophylactically treat an individual prior to theonset of a disorder characterized by or associated with aberrantexpression or activity of a polypeptide of the invention.

Another aspect of the invention provides methods for expression of anucleic acid or polypeptide of the invention or activity of apolypeptide of the invention in an individual to thereby selectappropriate therapeutic or prophylactic agents for that individual(referred to herein as “pharmacogenomics”). Pharmacogenomics allows forthe selection of agents (e.g., drugs) for therapeutic or prophylactictreatment of an individual based on the genotype of the individual(e.g., the genotype of the individual examined to determine the abilityof the individual to respond to a particular agent).

Yet another aspect of the invention pertains to monitoring the influenceof agents (e.g., drugs or other compounds) on the expression or activityof a polypeptide of the invention in clinical trials. These and otheragents are described in further detail in the following sections.

1. Diagnostic Assays

An exemplary method for detecting the presence or absence of apolypeptide or nucleic acid of the invention in a biological sampleinvolves obtaining a biological sample from a test subject andcontacting the biological sample with a compound or an agent capable ofdetecting a polypeptide or nucleic acid (e.g., mRNA, genomic DNA) of theinvention such that the presence of a polypeptide or nucleic acid of theinvention is detected in the biological sample. A preferred agent fordetecting mRNA or genomic DNA encoding a polypeptide of the invention isa labeled nucleic acid probe capable of hybridizing to mRNA or genomicDNA encoding a polypeptide of the invention. The nucleic acid probe canbe, for example, a full-length cDNA, such as the nucleic acid of SEQ IDNO:1, 2, 3, 13, 14, 19, 20, 31, 32, 39, 40, or 44, or a portion thereof,such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500nucleotides in length and sufficient to specifically hybridize understringent conditions to a mRNA or genomic DNA encoding a polypeptide ofthe invention. Other suitable probes for use in the diagnostic assays ofthe invention are described herein.

A preferred agent for detecting a polypeptide of the invention is anantibody capable of binding to a polypeptide of the invention,preferably an antibody with a detectable label. Antibodies can bepolyclonal, or more preferably, monoclonal. An intact antibody, or afragment thereof (e.g., Fab or F(ab′)₂) can be used. The term “labeled”,with regard to the probe or antibody, is intended to encompass directlabeling of the probe or antibody by coupling (i.e., physically linking)a detectable substance to the probe or antibody, as well as indirectlabeling of the probe or antibody by reactivity with another reagentthat is directly labeled. Examples of indirect labeling includedetection of a primary antibody using a fluorescently labeled secondaryantibody and end-labeling of a DNA probe with biotin such that it can bedetected with fluorescently labeled streptavidin. The term “biologicalsample” is intended to include tissues, cells and biological fluidsisolated from a subject, as well as tissues, cells and fluids presentwithin a subject. That is, the detection method of the invention can beused to detect mRNA, protein, or genomic DNA in a biological sample invitro as well as in vivo. For example, in vitro techniques for detectionof mRNA include Northern hybridizations and in situ hybridizations. Invitro techniques for detection of a polypeptide of the invention includeenzyme linked immunosorbent assays (ELISAs), Western blots,immunoprecipitations and immunofluorescence. In vitro techniques fordetection of genomic DNA include Southern hybridizations. Furthermore,in vivo techniques for detection of a polypeptide of the inventioninclude introducing into a subject a labeled antibody directed againstthe polypeptide. For example, the antibody can be labeled with aradioactive marker whose presence and location in a subject can bedetected by standard imaging techniques.

In one embodiment, the biological sample contains protein molecules fromthe test subject. Alternatively, the biological sample can contain mRNAmolecules from the test subject or genomic DNA molecules from the testsubject. A preferred biological sample is a peripheral blood leukocytesample isolated by conventional means from a subject.

In another embodiment, the methods further involve obtaining a controlbiological sample from a control subject, contacting the control samplewith a compound or agent capable of detecting a polypeptide of theinvention or mRNA or genomic DNA encoding a polypeptide of theinvention, such that the presence of the polypeptide or mRNA or genomicDNA encoding the polypeptide is detected in the biological sample, andcomparing the presence of the polypeptide or mRNA or genomic DNAencoding the polypeptide in the control sample with the presence of thepolypeptide or mRNA or genomic DNA encoding the polypeptide in the testsample.

The invention also encompasses kits for detecting the presence of apolypeptide or nucleic acid of the invention in a biological sample (atest sample). Such kits can be used to determine if a subject issuffering from or is at increased risk of developing a disorderassociated with aberrant expression of a polypeptide of the invention(e.g., a proliferative disorder, e.g., psoriasis or cancer). Forexample, the kit can comprise a labeled compound or agent capable ofdetecting the polypeptide or mRNA encoding the polypeptide in abiological sample and means for determining the amount of thepolypeptide or mRNA in the sample (e.g., an antibody which binds thepolypeptide or an oligonucleotide probe which binds to DNA or mRNAencoding the polypeptide). Kits can also include instructions forobserving that the tested subject is suffering from or is at risk ofdeveloping a disorder associated with aberrant expression of thepolypeptide if the amount of the polypeptide or mRNA encoding thepolypeptide is above or below a normal level.

For antibody-based kits, the kit can comprise, for example: (1) a firstantibody (e.g., attached to a solid support) which binds to apolypeptide of the invention; and, optionally, (2) a second, differentantibody which binds to either the polypeptide or the first antibody andis conjugated to a detectable agent.

For oligonucleotide-based kits, the kit can comprise, for example: (1)an oligonucleotide, e.g., a detectably labeled oligonucleotide, whichhybridizes to a nucleic acid sequence encoding a polypeptide of theinvention or (2) a pair of primers useful for amplifying a nucleic acidmolecule encoding a polypeptide of the invention. The kit can alsocomprise, e.g., a buffering agent, a preservative, or a proteinstabilizing agent. The kit can also comprise components necessary fordetecting the detectable agent (e.g., an enzyme or a substrate). The kitcan also contain a control sample or a series of control samples whichcan be assayed and compared to the test sample contained. Each componentof the kit is usually enclosed within an individual container and all ofthe various containers are within a single package along withinstructions for observing whether the tested subject is suffering fromor is at risk of developing a disorder associated with aberrantexpression of the polypeptide.

2. Prognostic Assays

The methods described herein can furthermore be utilized as diagnosticor prognostic assays to identify subjects having or at risk ofdeveloping a disease or disorder associated with aberrant expression oractivity of a polypeptide of the invention. For example, the assaysdescribed herein, such as the preceding diagnostic assays or thefollowing assays, can be utilized to identify a subject having or atrisk of developing a disorder associated with aberrant expression oractivity of a polypeptide of the invention, e.g., an immunologicdisorder, e.g., asthma, anaphylaxis, or atopic dermatitis.Alternatively, the prognostic assays can be utilized to identify asubject having or at risk for developing such a disease or disorder.Thus, the present invention provides a method in which a test sample isobtained from a subject and a polypeptide or nucleic acid (e.g., mRNA,genomic DNA) of the invention is detected, wherein the presence of thepolypeptide or nucleic acid is diagnostic for a subject having or atrisk of developing a disease or disorder associated with aberrantexpression or activity of the polypeptide. As used herein, a “testsample” refers to a biological sample obtained from a subject ofinterest. For example, a test sample can be a biological fluid (e.g.,serum), cell sample, or tissue.

Furthermore, the prognostic assays described herein can be used todetermine whether a subject can be administered an agent (e.g., anagonist, antagonist, peptidomimetic, protein, peptide, nucleic acid,small molecule, or other drug candidate) to treat a disease or disorderassociated with aberrant expression or activity of a polypeptide of theinvention. For example, such methods can be used to determine whether asubject can be effectively treated with a specific agent or class ofagents (e.g., agents of a type which decrease activity of thepolypeptide). Thus, the present invention provides methods fordetermining whether a subject can be effectively treated with an agentfor a disorder associated with aberrant expression or activity of apolypeptide of the invention in which a test sample is obtained and thepolypeptide or nucleic acid encoding the polypeptide is detected (e.g.,wherein the presence of the polypeptide or nucleic acid is diagnosticfor a subject that can be administered the agent to treat a disorderassociated with aberrant expression or activity of the polypeptide).

The methods of the invention can also be used to detect genetic lesionsor mutations in a gene of the invention, thereby determining if asubject with the lesioned gene is at risk for a disorder characterizedaberrant expression or activity of a polypeptide of the invention. Inpreferred embodiments, the methods include detecting, in a sample ofcells from the subject, the presence or absence of a genetic lesion ormutation characterized by at least one of an alteration affecting theintegrity of a gene encoding the polypeptide of the invention, or themis-expression of the gene encoding the polypeptide of the invention.For example, such genetic lesions or mutations can be detected byascertaining the existence of at least one of: 1) a deletion of one ormore nucleotides from the gene; 2) an addition of one or morenucleotides to the gene; 3) a substitution of one or more nucleotides ofthe gene; 4) a chromosomal rearrangement of the gene; 5) an alterationin the level of a messenger RNA transcript of the gene; 6) an aberrantmodification of the gene, such as of the methylation pattern of thegenomic DNA; 7) the presence of a non-wild type splicing pattern of amessenger RNA transcript of the gene; 8) a non-wild type level of a theprotein encoded by the gene; 9) an allelic loss of the gene; and 10) aninappropriate post-translational modification of the protein encoded bythe gene. As described herein, there are a large number of assaytechniques known in the art which can be used for detecting lesions in agene.

In certain embodiments, detection of the lesion involves the use of aprobe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Pat.Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) Proc.Natl. Acad. Sci. USA 91:360-364), the latter of which can beparticularly useful for detecting point mutations in a gene (see, e.g.,Abravaya et al. (1995) Nucleic Acids Res. 23:675-682). This method caninclude the steps of collecting a sample of cells from a patient,isolating nucleic acid (e.g., genomic, mRNA or both) from the cells ofthe sample, contacting the nucleic acid sample with one or more primerswhich specifically hybridize to the selected gene under conditions suchthat hybridization and amplification of the gene (if present) occurs,and detecting the presence or absence of an amplification product, ordetecting the size of the amplification product and comparing the lengthto a control sample. It is anticipated that PCR and/or LCR may bedesirable to use as a preliminary amplification step in conjunction withany of the techniques used for detecting mutations described herein.

Alternative amplification methods include: self sustained sequencereplication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh, et al. (1989)Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi etal. (1988) Bio/Technology 6:1197), or any other nucleic acidamplification method, followed by the detection of the amplifiedmolecules using techniques well known to those of skill in the art.These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

In an alternative embodiment, mutations in a selected gene from a samplecell can be identified by alterations in restriction enzyme cleavagepatterns. For example, sample and control DNA is isolated, amplified(optionally), digested with one or more restriction endonucleases, andfragment length sizes are determined by gel electrophoresis andcompared. Differences in fragment length sizes between sample andcontrol DNA indicates mutations in the sample DNA. Moreover, the use ofsequence specific ribozymes (see, e.g., U.S. Pat. No. 5,498,531) can beused to score for the presence of specific mutations by development orloss of a ribozyme cleavage site.

In other embodiments, genetic mutations can be identified by hybridizinga sample and control nucleic acids, e.g., DNA or RNA, to high densityarrays containing hundreds or thousands of oligonucleotides probes(Cronin et al. (1996) Human Mutation 7:244-255; Kozal et al. (1996)Nature Medicine 2:753-759). For example, genetic mutations can beidentified in two-dimensional arrays containing light-generated DNAprobes as described in Cronin et al., supra. Briefly, a firsthybridization array of probes can be used to scan through long stretchesof DNA in a sample and control to identify base changes between thesequences by making linear arrays of sequential overlapping probes. Thisstep allows the identification of point mutations. This step is followedby a second hybridization array that allows the characterization ofspecific mutations by using smaller, specialized probe arrayscomplementary to all variants or mutations detected. Each mutation arrayis composed of parallel probe sets, one complementary to the wild-typegene and the other complementary to the mutant gene.

In yet another embodiment, any of a variety of sequencing reactionsknown in the art can be used to directly sequence the selected gene anddetect mutations by comparing the sequence of the sample nucleic acidswith the corresponding wild-type (control) sequence. Examples ofsequencing reactions include those based on techniques developed byMaxim and Gilbert ((1977) Proc. Natl. Acad. Sci. USA 74:560) or Sanger((1977) Proc. Natl. Acad. Sci. USA 74:5463). It is also contemplatedthat any of a variety of automated sequencing procedures can be utilizedwhen performing the diagnostic assays ((1995) Bio/Techniques 19:448),including sequencing by mass spectrometry (see, e.g., PCT PublicationNo. WO 94/16101; Cohen et al. (1996) Adv. Chromatogr. 36:127-162; andGriffin et al. (1993) Appl. Biochem. Biotechnol. 38:147-159).

Other methods for detecting mutations in a selected gene include methodsin which protection from cleavage agents is used to detect mismatchedbases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science230:1242). In general, the technique of “mismatch cleavage” entailsproviding heteroduplexes formed by hybridizing (labeled) RNA or DNAcontaining the wild-type sequence with potentially mutant RNA or DNAobtained from a tissue sample. The double-stranded duplexes are treatedwith an agent which cleaves single-stranded regions of the duplex suchas which will exist due to basepair mismatches between the control andsample strands. RNA/DNA duplexes can be treated with RNase to digestmismatched regions, and DNA/DNA hybrids can be treated with S1 nucleaseto digest mismatched regions.

In other embodiments, either DNA/DNA or RNA/DNA duplexes can be treatedwith hydroxylamine or osmium tetroxide and with piperidine in order todigest mismatched regions. After digestion of the mismatched regions,the resulting material is then separated by size on denaturingpolyacrylamide gels to determine the site of mutation. See, e.g., Cottonet al. (1988) Proc. Natl. Acad. Sci. USA 85:4397; Saleeba et al. (1992)Methods Enzymol. 217:286-295. In a preferred embodiment, the control DNAor RNA can be labeled for detection.

In still another embodiment, the mismatch cleavage reaction employs oneor more proteins that recognize mismatched base pairs in double-strandedDNA (so called “DNA mismatch repair” enzymes) in defined systems fordetecting and mapping point mutations in cDNAs obtained from samples ofcells. For example, the mutY enzyme of E. coli cleaves A at G/Amismatches and the thymidine DNA glycosylase from HeLa cells cleaves Tat G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662).According to an exemplary embodiment, a probe based on a selectedsequence, e.g., a wild-type sequence, is hybridized to a cDNA or otherDNA product from a test cell(s). The duplex is treated with a DNAmismatch repair enzyme, and the cleavage products, if any, can bedetected from electrophoresis protocols or the like. See, e.g., U.S.Pat. No. 5,459,039.

In other embodiments, alterations in electrophoretic mobility will beused to identify mutations in genes. For example, single strandconformation polymorphism (SSCP) may be used to detect differences inelectrophoretic mobility between mutant and wild type nucleic acids(Orita et al. (1989) Proc. Natl. Acad. Sci. USA 86:2766; see also Cotton(1993) Mutat. Res. 285:125-144; Hayashi (1992) Genet. Anal. Tech. Appl.9:73-79). Single-stranded DNA fragments of sample and control nucleicacids will be denatured and allowed to renature. The secondary structureof single-stranded nucleic acids varies according to sequence, and theresulting alteration in electrophoretic mobility enables the detectionof even a single base change. The DNA fragments may be labeled ordetected with labeled probes. The sensitivity of the assay may beenhanced by using RNA (rather than DNA), in which the secondarystructure is more sensitive to a change in sequence. In a preferredembodiment, the subject method utilizes heteroduplex analysis toseparate double stranded heteroduplex molecules on the basis of changesin electrophoretic mobility (Keen et al. (1991) Trends Genet. 7:5).

In yet another embodiment, the movement of mutant or wild-type fragmentsin polyacrylamide gels containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (DGGE) (Myers et al.(1985) Nature 313:495). When DGGE is used as the method of analysis, DNAwill be modified to insure that it does not completely denature, forexample by adding a GC clamp of approximately 40 bp of high-meltingGC-rich DNA by PCR. In a further embodiment, a temperature gradient isused in place of a denaturing gradient to identify differences in themobility of control and sample DNA (Rosenbaum and Reissner (1987)Biophys. Chem. 265:12753).

Examples of other techniques for detecting point mutations include, butare not limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension. For example,oligonucleotide primers may be prepared in which the known mutation isplaced centrally and then hybridized to target DNA under conditionswhich permit hybridization only if a perfect match is found (Saiki etal. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl. Acad. Sci.USA 86:6230). Such allele specific oligonucleotides are hybridized toPCR amplified target DNA or a number of different mutations when theoligonucleotides are attached to the hybridizing membrane and hybridizedwith labeled target DNA.

Alternatively, allele specific amplification technology which depends onselective PCR amplification may be used in conjunction with the instantinvention. Oligonucleotides used as primers for specific amplificationmay carry the mutation of interest in the center of the molecule (sothat amplification depends on differential hybridization) (Gibbs et al.(1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3′ end of oneprimer where, under appropriate conditions, mismatch can prevent orreduce polymerase extension (Prossner (1993) Tibtech 11:238). Inaddition, it may be desirable to introduce a novel restriction site inthe region of the mutation to create cleavage-based detection (Gaspariniet al. (1992) Mol. Cell Probes 6:1). It is anticipated that in certainembodiments amplification may also be performed using Taq ligase foramplification (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189). In suchcases, ligation will occur only if there is a perfect match at the 3′end of the 5′ sequence making it possible to detect the presence of aknown mutation at a specific site by looking for the presence or absenceof amplification.

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one probe nucleic acidor antibody reagent described herein, which may be conveniently used,e.g., in clinical settings to diagnose patients exhibiting symptoms orfamily history of a disease or illness involving a gene encoding apolypeptide of the invention. Furthermore, any cell type or tissue,e.g., chondrocytes, in which the polypeptide of the invention isexpressed may be utilized in the prognostic assays described herein.

3. Pharmacogenomics

Agents, or modulators which have a stimulatory or inhibitory effect onactivity or expression of a polypeptide of the invention as identifiedby a screening assay described herein can be administered to individualsto treat (prophylactically or therapeutically) disorders associated withaberrant activity of the polypeptide. In conjunction with suchtreatment, the pharmacogenomics (i.e., the study of the relationshipbetween an individual's genotype and that individual's response to aforeign compound or drug) of the individual may be considered.Differences in metabolism of therapeutics can lead to severe toxicity ortherapeutic failure by altering the relation between dose and bloodconcentration of the pharmacologically active drug. Thus, thepharmacogenomics of the individual permits the selection of effectiveagents (e.g., drugs) for prophylactic or therapeutic treatments based ona consideration of the individual's genotype. Such pharmacogenomics canfurther be used to determine appropriate dosages and therapeuticregimens. Accordingly, the activity of a polypeptide of the invention,expression of a nucleic acid of the invention, or mutation content of agene of the invention in an individual can be determined to therebyselect appropriate agent(s) for therapeutic or prophylactic treatment ofthe individual.

Pharmacogenomics deals with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, e.g., Linder (1997) Clin. Chem.43(2):254-266. In general, two types of pharmacogenetic conditions canbe differentiated. Genetic conditions transmitted as a single factoraltering the way drugs act on the body are referred to as “altered drugaction.” Genetic conditions transmitted as single factors altering theway the body acts on drugs are referred to as “altered drug metabolism”.These pharmacogenetic conditions can occur either as rare defects or aspolymorphisms. For example, glucose-6-phosphate dehydrogenase deficiency(G6PD) is a common inherited enzymopathy in which the main clinicalcomplication is haemolysis after ingestion of oxidant drugs(anti-malarials, sulfonamides, analgesics, nitrofurans) and consumptionof fava beans.

As an illustrative embodiment, the activity of drug metabolizing enzymesis a major determinant of both the intensity and duration of drugaction. The discovery of genetic polymorphisms of drug metabolizingenzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymesCYP2D6 and CYP2C19) has provided an explanation as to why some patientsdo not obtain the expected drug effects or show exaggerated drugresponse and serious toxicity after taking the standard and safe dose ofa drug. These polymorphisms are expressed in two phenotypes in thepopulation, the extensive metabolizer (EM) and poor metabolizer (PM).The prevalence of PM is different among different populations. Forexample, the gene coding for CYP2D6 is highly polymorphic and severalmutations have been identified in PM, which all lead to the absence offunctional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quitefrequently experience exaggerated drug response and side effects whenthey receive standard doses. If a metabolite is the active therapeuticmoiety, a PM will show no therapeutic response, as demonstrated for theanalgesic effect of codeine mediated by its CYP2D6-formed metabolitemorphine. The other extreme are the so called ultra-rapid metabolizerswho do not respond to standard doses. Recently, the molecular basis ofultra-rapid metabolism has been identified to be due to CYP2D6 geneamplification.

Thus, the activity of a polypeptide of the invention, expression of anucleic acid encoding the polypeptide, or mutation content of a geneencoding the polypeptide in an individual can be determined to therebyselect appropriate agent(s) for therapeutic or prophylactic treatment ofthe individual. In addition, pharmacogenetic studies can be used toapply genotyping of polymorphic alleles encoding drug-metabolizingenzymes to the identification of an individual's drug responsivenessphenotype. This knowledge, when applied to dosing or drug selection, canavoid adverse reactions or therapeutic failure and thus enhancetherapeutic or prophylactic efficiency when treating a subject with amodulator of activity or expression of the polypeptide, such as amodulator identified by one of the exemplary screening assays describedherein.

4. Monitoring of Effects During Clinical Trials

Monitoring the influence of agents (e.g., drugs, compounds) on theexpression or activity of a polypeptide of the invention (e.g., theability to modulate aberrant cell proliferation chemotaxis, and/ordifferentiation) can be applied not only in basic drug screening, butalso in clinical trials. For example, the effectiveness of an agent, asdetermined by a screening assay as described herein, to increase geneexpression, protein levels or protein activity, can be monitored inclinical trials of subjects exhibiting decreased gene expression,protein levels, or protein activity. Alternatively, the effectiveness ofan agent, as determined by a screening assay, to decrease geneexpression, protein levels or protein activity, can be monitored inclinical trials of subjects exhibiting increased gene expression,protein levels, or protein activity. In such clinical trials, expressionor activity of a polypeptide of the invention and preferably, that ofother polypeptide that have been implicated in for example, a cellularproliferation disorder, can be used as a marker of the immuneresponsiveness of a particular cell.

For example, and not by way of limitation, genes, including those of theinvention, that are modulated in cells by treatment with an agent (e.g.,compound, drug or small molecule) which modulates activity or expressionof a polypeptide of the invention (e.g., as identified in a screeningassay described herein) can be identified. Thus, to study the effect ofagents on cellular proliferation disorders, for example, in a clinicaltrial, cells can be isolated and RNA prepared and analyzed for thelevels of expression of a gene of the invention and other genesimplicated in the disorder. The levels of gene expression (i.e., a geneexpression pattern) can be quantified by Northern blot analysis orRT-PCR, as described herein, or alternatively by measuring the amount ofprotein produced, by one of the methods as described herein, or bymeasuring the levels of activity of a gene of the invention or othergenes. In this way, the gene expression pattern can serve as a marker,indicative of the physiological response of the cells to the agent.Accordingly, this response state may be determined before, and atvarious points during, treatment of the individual with the agent.

In a preferred embodiment, the present invention provides a method formonitoring the effectiveness of treatment of a subject with an agent(e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleicacid, small molecule, or other drug candidate identified by thescreening assays described herein) comprising the steps of (i) obtaininga pre-administration sample from a subject prior to administration ofthe agent; (ii) detecting the level of the polypeptide or nucleic acidof the invention in the preadministration sample; (iii) obtaining one ormore post-administration samples from the subject; (iv) detecting thelevel the of the polypeptide or nucleic acid of the invention in thepost-administration samples; (v) comparing the level of the polypeptideor nucleic acid of the invention in the pre-administration sample withthe level of the polypeptide or nucleic acid of the invention in thepost-administration sample or samples; and (vi) altering theadministration of the agent to the subject accordingly. For example,increased administration of the agent may be desirable to increase theexpression or activity of the polypeptide to higher levels thandetected, i.e., to increase the effectiveness of the agent.Alternatively, decreased administration of the agent may be desirable todecrease expression or activity of the polypeptide to lower levels thandetected, i.e., to decrease the effectiveness of the agent.

C. Methods of Treatment

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a disorderor having a disorder associated with aberrant expression or activity ofa polypeptide of the invention. For example, disorders characterized byaberrant expression or activity of the polypeptides of the inventioninclude immunologic disorders. In addition, the nucleic acids,polypeptides, and modulators thereof of the invention can be used totreat immunologic diseases and disorders, including but not limited to,allergic disorders (e.g., anaphylaxis and allergic asthma) andinflammatory disorders (e.g., atopic dermatitis). Polypeptides of theinvention can also treat diseases associated with bacterial infection(e.g., tuberculosis, e.g., pulmonary tuberculosis), inflammatoryarthropathy, and bone and cartilage degenerative diseases and disorders(e.g., arthritis, e.g., rheumatoid arthritis), as well as otherdisorders described herein.

1. Prophylactic Methods

In one aspect, the invention provides a method for preventing in asubject, a disease or condition associated with an aberrant expressionor activity of a polypeptide of the invention, by administering to thesubject an agent which modulates expression or at least one activity ofthe polypeptide. Subjects at risk for a disease which is caused orcontributed to by aberrant expression or activity of a polypeptide ofthe invention can be identified by, for example, any or a combination ofdiagnostic or prognostic assays as described herein. Administration of aprophylactic agent can occur prior to the manifestation of symptomscharacteristic of the aberrancy, such that a disease or disorder isprevented or, alternatively, delayed in its progression. Depending onthe type of aberrancy, for example, an agonist or antagonist agent canbe used for treating the subject. For example, an antagonist of a TANGO240 protein may be used to treat an arthropathic disorder, e.g.,rheumatoid arthritis. The appropriate agent can be determined based onscreening assays described herein.

2. Therapeutic Methods

Another aspect of the invention pertains to methods of modulatingexpression or activity of a polypeptide of the invention for therapeuticpurposes. The modulatory method of the invention involves contacting acell with an agent that modulates one or more of the activities of thepolypeptide. An agent that modulates activity can be an agent asdescribed herein, such as a nucleic acid or a protein, anaturally-occurring cognate ligand of the polypeptide, a peptide, apeptidomimetic, or other small molecule. In one embodiment, the agentstimulates one or more of the biological activities of the polypeptide.Examples of such stimulatory agents include the active polypeptide ofthe invention and a nucleic acid molecule encoding the polypeptide ofthe invention that has been introduced into the cell. In anotherembodiment, the agent inhibits one or more of the biological activitiesof the polypeptide of the invention. Examples of such inhibitory agentsinclude antisense nucleic acid molecules and antibodies. Thesemodulatory methods can be performed in vitro (e.g., by culturing thecell with the agent) or, alternatively, in vivo (e.g., by administeringthe agent to a subject). As such, the present invention provides methodsof treating an individual afflicted with a disease or disordercharacterized by aberrant expression or activity of a polypeptide of theinvention. In one embodiment, the method involves administering an agent(e.g., an agent identified by a screening assay described herein), orcombination of agents that modulates (e.g., upregulates ordownregulates) expression or activity. In another embodiment, the methodinvolves administering a polypeptide of the invention or a nucleic acidmolecule of the invention as therapy to compensate for reduced oraberrant expression or activity of the polypeptide.

Stimulation of activity is desirable in situations in which activity orexpression is abnormally low or downregulated and/or in which increasedactivity is likely to have a beneficial effect. Conversely, inhibitionof activity is desirable in situations in which activity or expressionis abnormally high or upregulated and/or in which decreased activity islikely to have a beneficial effect.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication are hereby incorporated by reference.

Deposit of Clones

Clones containing cDNA molecules encoding TANGO 228, (clone EpT228),TANGO 240 (EpT240) and TANGO 243 (clone EpT243) were deposited with theAmerican Type Culture Collection, 10801 University Boulevard, Manassas,Va., 20110-2209, on Feb. 18, 1999 as Accession Number 207116, as part ofa composite deposit representing a mixture of three strains, eachcarrying one recombinant plasmid harboring a particular cDNA clone.

To distinguish the strains and isolate a strain harboring a particularcDNA clone, an aliquot of the mixture can be streaked out to singlecolonies on nutrient medium (e.g., LB plates) supplemented with 100μg/ml ampicillin, single colonies grown, and then plasmid DNA extractedusing a standard minipreparation procedure. Next, a sample of the DNAminipreparation can be digested with a combination of the restrictionenzymes Sal I and Not I and the resultant products resolved on a 0.8%agarose gel using standard DNA electrophoresis conditions. The digestliberates fragments as follows:

TANGO 228: 4.1 kb

TANGO 240: 2.2 kb

TANGO 243: 2.8 kb

The identity of the strains can be inferred from the fragmentsliberated.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1-22. (canceled)
 23. An isolated host cell which contains an isolatednucleic acid molecule which encodes a polypeptide comprising an aminoacid sequence which is at least 95% identical to the amino acid sequenceof SEQ ID NO:15, SEQ ID NO:16 or the amino acid sequence encoded by thecDNA insert of clone EpT240 which was deposited with ATCC as AccessionNumber 207116, wherein the polypeptide is capable of treating arthritis.24. The isolated host cell of claim 23, wherein the nucleic acidmolecule encodes a polypeptide comprising an amino acid sequence whichis at least 95% identical to the amino acid sequence of SEQ ID NO:15,wherein the polypeptide is capable of treating arthritis.
 25. Theisolated host cell of claim 23, wherein the nucleic acid moleculeencodes a polypeptide comprising an amino acid sequence which is atleast 95% identical to the amino acid sequence of SEQ ID NO:16, whereinthe polypeptide is capable of treating arthritis.
 26. The isolated hostcell of claim 23, wherein the nucleic acid molecule encodes apolypeptide comprising an amino acid sequence which is at least 95%identical to the amino acid sequence encoded by the cDNA insert of cloneEpT240 which was deposited with ATCC as Accession Number 207116, whereinthe polypeptide is capable of treating arthritis.
 27. The isolated hostcell of claim 23, wherein the nucleic acid molecule further comprisesvector nucleic acid sequences.
 28. The isolated host cell of claim 23,wherein the nucleic acid molecule further comprises nucleic acidsequences encoding a heterologous polypeptide.
 29. The isolated hostcell of claim 23, wherein the isolated host cell is an isolated humanhost cell.
 30. A method for producing a polypeptide selected from thegroup consisting of: a) a polypeptide comprising an amino acid sequencewhich is at least 95% identical to the amino acid sequence of SEQ IDNO:15, SEQ ID NO:16 or the amino acid sequence encoded by the cDNAinsert of clone EpT240 which was deposited with ATCC as Accession Number207116, wherein the polypeptide is capable of treating arthritis; and b)a polypeptide encoded by a nucleic acid molecule comprising a nucleotidesequence which is at least 95% identical to a nucleic acid comprisingthe nucleotide sequence of SEQ ID NO:13, SEQ ID NO:14, the cDNA insertof clone EpT240 which was deposited with ATCC as Accession Number207116, or the complement thereof, wherein the polypeptide is capable oftreating arthritis; comprising culturing the isolated host cell of claim23 under conditions in which the nucleic acid molecule is expressed. 31.The method of claim 30, wherein the polypeptide comprises an amino acidsequence which is at least 95% identical to the amino acid sequence ofSEQ ID NO:15, SEQ ID NO:16 or the amino acid sequence encoded by thecDNA insert of clone EpT240 which was deposited with ATCC as AccessionNumber 207116, wherein the polypeptide is capable of treating arthritis.32. The method of claim 30, wherein the polypeptide is encoded by anucleic acid molecule comprising a nucleotide sequence which is at least95% identical to a nucleic acid comprising the nucleotide sequence ofSEQ ID NO:13, SEQ ID NO:14, the cDNA insert of clone EpT240 which wasdeposited with ATCC as Accession Number 207116, or the complementthereof, wherein the polypeptide is capable of treating arthritis. 33.The method of claim 30, wherein the isolated host cell is an isolatedhuman host cell.
 34. An isolated host cell which contains an isolatednucleic acid molecule selected from the group consisting of: a) anucleic acid molecule comprising the nucleotide sequence of SEQ IDNO:13; b) a nucleic acid molecule comprising the nucleotide sequence ofSEQ ID NO:14; c) a nucleic acid molecule which encodes a polypeptidecomprising the amino acid sequence of SEQ ID NO:15; d) a nucleic acidmolecule which encodes a polypeptide comprising the amino acid sequenceof SEQ ID NO:16; and e) a nucleic acid molecule which encodes apolypeptide comprising the amino acid sequence encoded by the cDNAinsert of clone EpT240 which was deposited with ATCC as Accession Number207116.
 35. The isolated host cell of claim 34, wherein the nucleic acidmolecule further comprises vector nucleic acid sequences.
 36. Theisolated host cell of claim 34, wherein the nucleic acid moleculefurther comprises nucleic acid sequences encoding a heterologouspolypeptide.
 37. The isolated host cell of claim 34, wherein theisolated host cell is an isolated human host cell.
 38. A method forproducing a polypeptide selected from the group consisting of: a) apolypeptide comprising the amino acid sequence of SEQ ID NO:15, SEQ IDNO:16, or the amino acid sequence encoded by the cDNA insert of cloneEpT240 which was deposited with ATCC as Accession Number 207116; and b)a polypeptide encoded by a nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO:13, SEQ ID NO:14, the cDNA insert ofclone EpT240 which was deposited with ATCC as Accession Number 207116,or the complement thereof; comprising culturing the isolated host cellof claim 34 under conditions in which the nucleic acid molecule isexpressed.
 39. The method of claim 38, wherein the polypeptide comprisesthe amino acid sequence of SEQ ID NO:15.
 40. The method of claim 38,wherein the polypeptide comprises the amino acid sequence of SEQ IDNO:16.
 41. The method of claim 38, wherein the polypeptide is encoded bya nucleic acid molecule comprising the nucleotide sequence of SEQ IDNO:13.
 42. The method of claim 38, wherein the polypeptide is encoded bya nucleic acid molecule comprising the nucleotide sequence of SEQ IDNO:14.
 43. The method of claim 38, wherein the polypeptide is encoded bya nucleic acid molecule comprising the cDNA insert of clone EpT240 whichwas deposited with ATCC as Accession Number
 207116. 44. The method ofclaim 38, wherein the isolated host cell is an isolated human host cell.